Cysteine engineered fibronectin type III domain binding molecules

ABSTRACT

Cysteine engineered monospecific and bispecific EGFR and/or c-Met FN3 domain containing molecules comprising one or more free cysteine amino acids are prepared by mutagenizing a nucleic acid sequence of a parent molecule and replacing one or more amino acid residues by cysteine to encode the cysteine engineered FN3 domain containing monospecific or bispecific molecules; expressing the cysteine engineered FN3 domain containing molecules; and recovering the cysteine engineered FN3 domain containing molecule. Isolated cysteine engineered monospecific or bispecific FN3 domain containing molecules may be covalently attached to a detection label or a drug moiety and used therapeutically.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/352,776, filed 21 Jun. 2016. The entire contents of theaforementioned application are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to binding molecules engineered withcysteine residues and methods of making and using the same. Moreparticularly, the invention is directed to fibronectin type III (FN3)domain molecules that may bind to EGFR and/or c-Met that are cysteineengineered.

BACKGROUND OF THE INVENTION

Epidermal growth factor receptor (EGFR or ErbB1 or HER1) is atransmembrane glycoprotein of 170 kDa that is encoded by the c-erbB1proto-oncogene. EGFR is a member of the human epidermal growth factorreceptor (HER) family of receptor tyrosine kinases (RTK) which includesHER2 (ErbB2), HER3 (ErbB3) and HER4 (ErbB4). These RTKs share ahomologous structure that consists of a ligand-binding extracellulardomain (ECD), a single span transmembrane domain and an intracellulardomain that contain catalytic kinase domain and a C-terminal tail. EGFRsignaling is initiated by ligand binding followed by induction ofconformational change, dimerization and trans-autophosphorylation of thereceptor (Ferguson et al., Annu Rev Biophys, 37: 353-73, 2008) whichinitiates a signal transduction cascade that ultimately affects a widevariety of cellular functions, including cell proliferation andsurvival. Increases in expression or kinase activity of EGFR have beenlinked with a range of human cancers, making EGFR an attractive targetfor therapeutic intervention (Mendelsohn et al., Oncogene 19: 6550-6565,2000; Grünwald et al., J Natl Cancer Inst 95: 851-67, 2003; Mendelsohnet al., Semin Oncol 33: 369-85, 2006). Furthermore, increases in boththe EGFR gene copy number and protein expression have been associatedwith favorable responses to the EGFR tyrosine kinase inhibitor, IRESSA™(gefitinib), in non-small cell lung cancer (Hirsch et al., Ann Oncol18:752-60, 2007).

EGFR therapies include both small molecules and anti-EGFR antibodies,approved for treatment of colorectal cancer, pancreatic cancer, head andneck cancer, and non-small cell lung cancer (NSCLC) (Baselga andArteaga, J Clin Oncol 23:2445-2459 (20005; Gill et al., J Biol Chem,259:7755-7760, 1984; Goldstein et al., Clin Cancer Res, 1:131 1-1318;1995; Prewett et al., Clin Cancer Res, 4:2957-2966, 1998).

Efficacy of anti-EGFR therapies may depend on tumor type and EFGRmutation/amplification status in the tumor, and may result in skintoxicity (De Roock et al., Lancet Oncol 11:753-762, 2010; Linardou etal., Nat Rev Clin Oncol, 6: 352-366, 2009; Li and Perez-Soler, TargOncol 4: 107-119, 2009). EGFR tyrosine kinase inhibitors (TKI) arecommonly used as 2^(nd) line therapies for non small cell lung cancer(NSCLC), but often stop working within twelve months due to resistancepathways (Riely et al., Clin Cancer Res 12: 839-44, 2006).

c-Met encodes a tyrosine kinase receptor. It was first identified as aproto-oncogene in 1984 after it was found that treatment with acarcinogen resulted in a constitutively active fusion protein TPR-MET(Cooper et al., Nature 311:29-33, 1984). Activation of c-Met through itsligand HGF stimulates a plethora of cell processes including growth,motility, invasion, metastasis, epithelial-mesenchymal transition,angiogenesis/wound healing, and tissue regeneration (Christensen et al.,Cancer Lett 225:1-26, 2005; Peters and Adjei, Nat Rev Clin Oncol9:314-26, 2012). c-Met is synthesized as a single chain protein that isproteolytically cleaved into a 50 kDa alpha- and 140 kDa beta-subunitlinked by a disulphide bond (Ma et al., Cancer and Metastasis Reviews,22: 309-325, 2003). c-Met is structurally similar to other membranereceptors such as Ron and Sea and is comprised of an extracellularligand-binding domain, a transmembrane domain, and a cytoplasmic domain(containing the tyrosine kinase domain and a C-terminal tail region).The exact stoichiometry of HGF:c-Met binding is unclear, but it isgenerally believed that two HGF molecules bind to two c-Met moleculesleading to receptor dimerization and autophosphorylation at tyrosines1230, 1234, and 1235 (Stamos et al., The EMBO Journal 23: 2325-2335,2004). Ligand-independent c-Met autophosphorylation can also occur dueto gene amplification, mutation or receptor overexpression.

c-Met is frequently amplified, mutated or over-expressed in many typesof cancer including gastric, lung, colon, breast, bladder, head andneck, ovarian, prostate, thyroid, pancreatic, and CNS. Missensemutations typically localized to the kinase domain are commonly found inhereditary papillary renal carcinomas (PRCC) and in 13% of sporadicPRCCs (Schmidt et al., Oncogene 18: 2343-2350, 1999). In contrast, c-Metmutations localized to the semaphorin or juxtamembrane domains of c-Metare frequently found in gastric, head and neck, liver, ovarian, NSCLCand thyroid cancers (Ma et al., Cancer and Metastasis Reviews, 22:309-325, 2003; Sakakura et al., Chromosomes and Cancer, 1999.24:299-305). c-Met amplification has been detected in brain, colorectal,gastric, and lung cancers, often correlating with disease progression(Ma et al., Cancer and Metastasis Reviews, 22: 309-325, 2003). Up to 4%and 20% of non-small cell lung cancer (NSCLC) and gastric cancers,respectively, exhibit c-Met amplification (Sakakura et al., Chromosomesand Cancer, 1999. 24:299-305: Sierra and Tsao, Therapeutic Advances inMedical Oncology, 3:S21-35, 2011). Even in the absence of geneamplification, c-Met overexpression is frequently observed in lungcancer (Ichimura et al., Jpn J Cancer Res, 87:1063-9, 1996). Moreover,in clinical samples, nearly half of lung adenocarcinomas exhibited highlevels of c-Met and HGF, both of which correlated with enhanced tumorgrowth rate, metastasis and poor prognosis (Sierra and Tsao, TherapeuticAdvances in Medical Oncology, 3:S21-35, 2011; Siegfried et al., AnnThorac Surg 66: 1915-8, 1998).

Nearly 60% of all tumors that become resistant to EGFR tyrosine kinaseinhibitors increase c-Met expression, amplify c-Met, or increase itsonly known ligand, HGF (Turke et al., Cancer Cell, 17:77-88, 2010),suggesting the existence of a compensatory pathway for EGFR throughc-Met. c-Met amplification was first identified in cultured cells thatbecame resistant to gefinitib, an EGFR kinase inhibitor, and exhibitedenhanced survival through the Her3 pathway (Engelman et al., Science,316:1039-43, 2007). This was further validated in clinical samples wherenine of 43 patients with acquired resistance to either erlotinib orgefitinib exhibited c-Met amplification, compared to only two of 62untreated patients. Interestingly, four of the nine treated patientsalso acquired the EGFR activating mutation, T790M, demonstratingsimultaneous resistance pathways (Beat et al., Proc Natl Acad Sci USA,104:20932-7, 2007).

The individual roles of both EGFR and c-Met in cancer is now wellestablished, making these targets attractive for combination therapy.Both receptors signal through the same survival and anti-apoptoticpathways (ERK and AKT); thus, inhibiting the pair in combination maylimit the potential for compensatory pathway activation therebyimproving overall efficacy. Combination therapies targeting EGFR andc-Met are tested in clinical trials with Tarceva (erlotinib) incombination with anti-c-Met monovalent antibody for NSCL (Spigel et al.,2011 ASCO Annual Meeting Proceedings 2011, Journal of Clinical Oncology:Chicago, Ill. p. 7505) and Tarceva (erlotinib) in combination withARQ-197, a small molecule inhibitor of c-Met (Adjei et al., Oncologist,16:788-99, 2011). Combination therapies or bispecific anti-EGFR/c-Metmolecules have been disclosed for example in: Int. Pat. Publ. No.WO2008/127710, U.S. Pat. Publ. No. US2009/0042906, Int. Pat. Publ. No.WO2009/111691, Int. Pat. Publ. No. WO2009/126834, Int. Pat. Publ. No.WO2010/039248, Int. Pat. Publ. No. WO2010/115551.

Current small molecule and large molecule (i.e. antibody) approaches toantagonize EGFR and/or c-Met signaling pathways for therapy may besub-optimal due to possible lack of specificity with small molecules andtherefore potential off-target activity and dose-limiting toxicityencountered with small molecule inhibitors. Typical bivalent antibodiesmay result in clustering of membrane bound receptors and unwantedactivation of the downstream signaling pathways, and monovalentantibodies (half arms) pose significant complexity and cost to themanufacturing process.

Accordingly, the need exists for additional monospecific and bispecificEGFR and/or c-Met inhibitors that also have the additional capability ofconjugating cytotoxic drugs thus targeting these potent compounds to theEGFR/c-met-expressing tumor cells, enhancing the anti-tumor activity ofthese EGFR/c-Met inhibitors. While antibody drug conjugates exist in theart, conventional means of attaching a drug moiety generally leads to aheterogeneous mixture of molecules where the drug moieties are attachedat a number of sites on the antibody. For example, cytotoxic drugs havetypically been conjugated to antibodies through the often-numerouslysine residues of an antibody, generating a heterogeneous antibody-drugconjugate mixture. Depending on reaction conditions, the heterogeneousmixture typically contains a distribution of antibodies with from 0 toabout 8, or more, attached drug moieties. In addition, within eachsubgroup of conjugates with a particular integer ratio of drug moietiesto antibodies, is a potentially heterogeneous mixture where the drugmoiety is attached at various sites on the antibody. Analytical andpreparative methods are inadequate to separate and characterize theantibody-drug conjugate species molecules within the heterogeneousmixture resulting from a conjugation reaction. Antibodies are large,complex and structurally diverse biomolecules, often with many reactivefunctional groups. Their reactivities with linker reagents anddrug-linker intermediates are dependent on factors, such as pH,concentration, salt concentration, and co-solvents. Furthermore, themultistep conjugation process may be non-reproducible due todifficulties in controlling the reaction conditions and characterizingreactants and intermediates.

Chemical conjugation via cysteines present in antibodies has also beendemonstrated. However, engineering in cysteine thiol groups by themutation of various amino acid residues of a protein to cysteine aminoacids is potentially problematic, particularly in the case of unpaired(free Cys) residues or those that are relatively accessible for reactionor oxidation. Unpaired Cys residues on the surface of the protein canpair and oxidize to form intermolecular disulfides, and hence proteindimers or multimers. Disulfide dimer formation renders the new Cysunreactive for conjugation to a drug, ligand, or other label.Furthermore, if the protein oxidatively forms an intramoleculardisulfide bond between the newly engineered Cys and an existing Cysresidue, both Cys groups are unavailable for active site participationand interactions. In addition, the protein may be rendered inactive ornonspecific, by misfolding or loss of tertiary structure (Zhang et al(2002) Anal. Biochem. 311: 1-9).

Thus, a need exists for a molecule that can undergo homogeneous chemicalconjugation and avoid the issues faced by antibody conjugates.

SUMMARY OF THE INVENTION

The present invention provides an isolated cysteine engineeredfibronectin type III (FN3) domain comprising at least one cysteinesubstitution at a position selected from the group consisting ofresidues 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48,53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, and 93 of the FN3 domainbased on SEQ ID NO: 27, and the equivalent positions in related FN3domains. A cysteine substitution at a position in the domain or proteincomprises a replacement of the existing amino acid residue with acysteine residue.

The present invention also provides an isolated cysteine engineeredfibronectin type III (FN3) domain comprising the amino acid sequence ofSEQ ID NO: 27 with at least one cysteine substitution from the aminoacid sequence of SEQ ID NO: 27 and specifically binds epidermal growthfactor receptor (EGFR) and blocks binding of epidermal growth factor(EGF) to EGFR.

The present invention further provides an isolated cysteine engineeredfibronectin type III (FN3) domain comprising the amino acid sequence ofSEQ ID NO: 114 with at least one cysteine substitution from the aminoacid sequence of SEQ ID NO: 114, and specifically binds hepatocytegrowth factor receptor (c-Met) and blocks binding of hepatocyte growthfactor (HGF) to c-Met.

The present invention provides novel positions at which cysteinesubstitutions may be made to generate the cysteine engineered FN3domains. Said positions include one or more of residues 6, 8, 10, 11,14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64,70, 88, 89, 90, 91, or 93 of SEQ ID NOS: 11-114 and/or 122-137.

An aspect of the invention is a process to prepare the isolated cysteineengineered FN3 domains by mutagenizing a nucleic acid sequence of aparent FN3 domain by replacing one or more amino acid residues with acysteine residue to encode the cysteine engineered FN3 domain;expressing the cysteine engineered FN3 domain; and isolating thecysteine engineered FN3 domain.

Another aspect of the invention is a chemically-conjugated, isolatedcysteine engineered FN3 domain wherein the FN3 domain is covalentlyattached to a chemical reagent comprising a maleimide moiety.

Another embodiment of the invention is a chemically-conjugated, isolatedcysteine engineered FN3 domain that can inhibit the growth ofEGFR-overexpressing and/or c-Met-expressing tumor cell lines.

The present application also provides an isolated cysteine engineeredbispecific FN3 molecule comprising a first fibronectin type III (FN3)domain and a second FN3 domain, wherein the first and second FN3 domainscomprise cysteine substitutions at a position selected from the groupconsisting of residues 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41,45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, and 93,specifically binds epidermal growth factor receptor (EGFR) and blocksbinding of epidermal growth factor (EGF) to EGFR, and the second FN3domain specifically binds hepatocyte growth factor receptor (c-Met), andblocks binding of hepatocyte growth factor (HGF) to c-Met.

Another aspect of the invention is a chemically-conjugated, isolatedcysteine engineered bispecific molecule wherein the bispecific moleculeis covalently attached to a chemical reagent comprising a maleimidemoiety.

A further aspect of the invention is a process to prepare the isolatedcysteine engineered bispecific FN3 by mutagenizing a nucleic acidsequence of a parent FN3 bispecific molecule by replacing one or moreamino acid residues with cysteine residues to encode the cysteineengineered bispecific molecule; expressing the cysteine engineeredmolecule; and isolating the cysteine engineered bispecific molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Amino acid alignment of the EGFR-binding FN3 domains.The BC and FG loops are boxed at residues 22-28 and 75-86 of SEQ ID NO:18. Some variants include thermal stability improving L17A, N46K andE86I substitutions (residue numbering according to Tencon SEQ ID NO: 1).

FIG. 2. Cytotoxin/linker structures.

FIG. 3. Ribbon representation of the crystal structure of P54AR4-83v2protein (SEQ ID NO: 27). Final positions identified as tolerant ofcysteine substitutions are shown as sticks and colored solid black.Binding loops BC/FG are colored shaded gray.

FIG. 4. Sequence alignment of the Tencon27 scaffold (SEQ ID NO: 99) anda TCL14 library (SEQ ID NO: 100) having randomized C-CD-F-FG alternativesurface. The loop residues are boxed. Loops and strands are indicatedabove the sequences.

FIGS. 5A and 5B. Sequence alignment of the c-Met-binding FN3 domains.The C loop and the CD strand and the F loop and the FG strand are boxedand span residues 29-43 and 65-81.

FIG. 6. Inhibition of c-Met phosphorylation in H292 cells pre-treatedwith monospecific or bispecific FN3 domain containing molecules andstimulated with HGF is shown. Substantial increase in the potency of thebispecific EGFR/c-Met molecule (ECB1) was observed when compared to amonospecific c-Met-binding FN3 domain (P114AR5P74-A5, shown as A5 in theFigure) on its own or in combination with an EGFR-binding FN3 domain(P54AR4-83v2, shown as 83v2 in the Figure).

FIG. 7. Inhibition of EGFR and c-Met phosphorylation in cellspre-treated with monospecific or bispecific FN3 domain containingmolecules. In cell lines expressing high levels of EGFR, H292 (A) andH596 (B), anti-EGFR monospecific and bispecific FN3 domain containingmolecules are equally potent at decreasing EGFR phosphorylation. In celllines expressing low levels of EGFR relative to c-Met, H441 (C),bispecific EGFR/c-Met molecules improve the potency for inhibition ofEGFR phosphorylation compared to the monospecific EGFR-binding FN3domain alone. In cell lines with low levels of c-Met, relative to EGFR,H292 (D) and H596 (E), inhibition of c-Met phosphorylation issignificantly potentiated with bispecific EGFR/c-Met molecule, comparedto monospecific c-Met-binding FN3 domain only. Molecules used in thestudy were: bispecific ECB5 (shown as 17-A3 in the Figure), monospecificEGFR-binding FN3 domain P53A1R5-17 (shown as “17” in the Figure),bispecific EGFR/c-Met molecule ECB3 (shown as 83-H9 in the Figure), andmonospecific c-Met binding FN3 domain P114AR7P93-H9 (shown as H9 in theFigure).

FIG. 8. Pharmacodynamic signaling in tumors isolated from mice dosedwith bispecific EGFR/c-Met molecules for 6 h or 72 h is shown. Allmolecules significantly reduced c-Met, EGFR and ERK phosphorylation atboth 6 h and 72 h, the degree if inhibition was dependent on theaffinity of the FN3 domains to EGFR and/or c-Met. Bispecific moleculeswere generated by joining EGFR-binding FN3 domain with a high (83 isp54AR4-83v2) or medium (“17v2” in the Figure is P53A1R5-17v2) affinityto a c-Met-binding FN3 domain with high (“A3” in the Figure isP114AR7P94-A3) or medium (“A5” in the Figure is P114ARSP74-A5) affinity.

FIG. 9: Plasma (top) and tumor (bottom) accumulation of bispecificEGFR/cMet molecules of variable affinities linked to an albumin bindingdomain (ABD) are shown 6 h (left) and 72 h (right) after IP dosing. Sixhours after dosing, tumor accumulation is maximal in mice dosed with abispecific molecule harboring a medium affinity EGFR-binding FN3 domain(17v2) and high affinity c-Met binding domain (83v2). The bispecificmolecules incorporated high or medium affinity EGFR or c-Met binding FN3domains as follows: 83v2-A5-ABD (ECB18; high/medium for EGFR/cMet)83v2-A3-ABD (ECB38; high/high) 17v2-A5 (ECB28; medium/medium)17v2-A3-ABD (ECB39; medium/high). 83v2 refers to p54AR4-83v2; 17v2refers to p53A1R5-17v2; A3 refers to p114AR7P94-A3; A5 refers top114AR5P74-A5.

FIG. 10. H292-HGF tumor xenografts were implanted into SCID beige mice.When tumors reached an average volume of approximately 80 mm³, mice weredosed three times per week with bispecific EGFR/c-Met molecules (25mg/kg) or PBS vehicle. All bispecific molecules reduced tumor growth,the tumor growth inhibition (TGI) being dependent on the affinities ofthe molecules for c-Met and EGFR. (high EGFR-high cMet refers top54AR4-83v2-p114AR7P94-A3 (ECB38); high EGFR-med cMet refers top54AR4-83v2-p114AR5P74-A5 (ECB18); med EGFR-high cMet refers top53A1R5-17v2-p114AR7P94-A3 (ECB39); med EGFR-med-cMet refers top53A1R5-17-p114AR5P74-A 5 (ECB28)).

FIG. 11. H292-HGF tumor xenografts were implanted into SCID beige miceand they were treated with different therapies. The anti-tumor activityof the therapies is shown. (bispecific EGFR/c-Met molecule refers top54AR4-83v2-p114AR7P94-A3-ABD (ECB38); the other therapies arecrizotinib, erlotinib, cetuximab, and the combination of crizotinib anderlotinib).

DETAILED DESCRIPTION OF THE INVENTION

The term “fibronectin type III (FN3) domain” (FN3 domain) as used hereinrefers to a domain occurring frequently in proteins includingfibronectins, tenascin, intracellular cytoskeletal proteins, cytokinereceptors and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad SciUSA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993;Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3domains are the 15 different FN3 domains present in human tenascin C,the 15 different FN3 domains present in human fibronectin (FN), andnon-natural synthetic FN3 domains as described for example in U.S. Pat.Publ. No. 2010/0216708. Individual FN3 domains are referred to by domainnumber and protein name, e.g., the 3^(rd) FN3 domain of tenascin (TN3),or the 10^(th) FN3 domain of fibronectin (FN10).

The term “substituting” or “substituted” or “mutating” or “mutated” asused herein refers to altering, deleting of inserting one or more aminoacids or nucleotides in a polypeptide or polynucleotide sequence togenerate a variant of that sequence.

The term “randomizing” or “randomized” or “diversified” or“diversifying” as used herein refers to making at least onesubstitution, insertion or deletion in a polynucleotide or polypeptidesequence.

“Variant” as used herein refers to a polypeptide or a polynucleotidethat differs from a reference polypeptide or a reference polynucleotideby one or more modifications for example, substitutions, insertions ordeletions.

The term “specifically binds” or “specific binding” as used hereinrefers to the ability of the FN3 domain of the invention to bind to apredetermined antigen with a dissociation constant (K_(D)) of 1×10⁻⁶ Mor less, for example 1×10⁻⁷ M or less, 1×10⁻⁸ M or less, 1×10⁻⁹ M orless, 1×10⁻¹⁰ M or less, 1×10⁻¹¹ M or less, 1×10⁻¹² M or less, or1×10⁻¹³ M or less. Typically the FN3 domain of the invention binds to apredetermined antigen (i.e. EGFR or c-Met) with a K_(D) that is at leastten fold less than its K_(D) for a nonspecific antigen (for example BSAor casein) as measured by surface plasmon resonance using for example aProteon Instrument (BioRad). Thus, a bispecific EGFR/c-Met FN3 domaincontaining molecule of the invention specifically binds to each EGFR andc-Met with a binding affinity (K_(D)) of at least 1×10⁻⁶ M or less forboth EGFR and c-Met. The isolated FN3 domain of the invention thatspecifically binds to a predetermined antigen may, however, havecross-reactivity to other related antigens, for example to the samepredetermined antigen from other species (homologs).

The term “library” refers to a collection of variants. The library maybe composed of polypeptide or polynucleotide variants.

The term “stability” as used herein refers to the ability of a moleculeto maintain a folded state under physiological conditions such that itretains at least one of its normal functional activities, for example,binding to a predetermined antigen such as EGFR or c-Met.

“Epidermal growth factor receptor” or “EGFR” as used here refers to thehuman EGFR (also known as HER-1 or Erb-B1 (Ullrich et al., Nature309:418-425, 1984) having the sequence shown in SEQ ID NO: 73 and inGenBank accession number NP_005219, as well as naturally-occurringvariants thereof. Such variants include the well known EGFRvIII andother alternatively spliced variants (e.g., as identified by SwissProtAccession numbers P00533-1, P00533-2, P00533-3, P00533-4), variantsGLN-98, ARG-266, Lys-521, ILE-674, GLY-962, and PRO-988 (Livingston etal., NIEHS-SNPs, environmental genome project, NIEHS ES15478).

“EGFR ligand” as used herein encompasses all (e.g., physiological)ligands for EGFR, including EGF, TGF-α, heparin binding EGF (HB-EGF),amphiregulin (AR), and epiregulin (EPI).

“Epidermal growth factor” (EGF) as used herein refers to the well known53 amino acid human EGF having an amino acid sequence shown in SEQ IDNO: 74.

“Hepatocyte growth factor receptor” or “c-Met” as used herein refers tothe human c-Met having the amino acid sequence shown in SEQ ID NO: 101or in GenBank Accession No: NP_001120972 and natural variants thereof.

“Hepatocyte growth factor” (HGF) as used herein refers to the well knownhuman HGF having the amino acid sequence shown in SEQ ID NO: 102 whichis cleaved to form a dimer of an alpha and beta chain linked by adisulfide bond.

“Blocks binding” or “inhibits binding”, as used herein interchangeablyrefers to the ability of the FN3 domains of the invention of thebispecific EGFR/c-Met FN3 domain containing molecule to block or inhibitbinding of the EGFR ligand such as EGF to EGFR and/or HGF to c-Met, andencompass both partial and complete blocking/inhibition. Theblocking/inhibition of EGFR ligand such as EGF to EGFR and/or HGF toc-Met by the FN3 domain or the bispecific EGFR/c-Met FN3 domaincontaining molecule of the invention reduces partially or completely thenormal level of EGFR signaling and/or c-Met signaling when compared tothe EGFR ligand binding to EGFR and/or HGF binding to c-Met withoutblocking or inhibition. The FN3 domain or the bispecific EGFR/c-Met FN3domain containing molecule of the invention “blocks binding” of the EGFRligand such as EGF to EGFR and/or HGF to c-Met when the inhibition is atleast 30%, 35%, 40%/0, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%/0, 85%,90%, 91%, 92%/0, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Inhibitionof binding can be measured using well known methods, for example bymeasuring inhibition of binding of biotinylated EGF on EGFR expressingA431 cells exposed to the FN3 domain or the bispecific EGFR/c-Met FN3domain containing molecule of the invention using FACS, and usingmethods described herein, or measuring inhibition of binding ofbiotinylated HGF on c-Met extracellular domain using well known methodsand methods described herein.

The term “EGFR signaling” refers to signal transduction induced by EGFRligand binding to EGFR resulting in autophosphorylation of at least onetyrosine residue in the EGFR. An exemplary EGFR ligand is EGF.

“Neutralizes EGFR signaling” as used herein refers to the ability of theFN3 domain of the invention to inhibit EGFR signaling induced by EGFRligand such as EGF by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100%.

The term “c-Met signaling” refers to signal transduction induced by HGFbinding to c-Met resulting in autophosphorylation of at least onetyrosine residue in the c-Met. Typically at least one tyrosine residueat positions 1230, 1234 or 1235 is autophosphorylated upon HGF binding.

“Neutralizes c-Met signaling” as used herein refers to the ability ofthe FN3 domain of the invention to inhibit c-Met signaling induced byHGF by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

“Overexpress, “overexpressed” and “overexpressing” as used hereininterchangeably refer to a cancer or malignant cell that has measurablyhigher levels of EGFR and/or c-Met on the surface compared to a normalcell of the same tissue type. Such overexpression may be caused by geneamplification or by increased transcription or translation. EGFR and/orc-Met expression and overexpression can be measured using well knowassays using for example ELISA, immunofluorescence, flow cytometry orradioimmunoassay on live or lysed cells. Alternatively, or additionally,levels of EGFR and/or c-Met-encoding nucleic acid molecules may bemeasured in the cell for example using fluorescent in situhybridization, Southern blotting, or PCR techniques. EGFR and/or c-Metis overexpressed when the level of EGFR and/or c-Met on the surface ofthe cell is at least 1.5-fold higher when compared to the normal cell.

“Tencon” as used herein refers to the synthetic fibronectin type III(FN3) domain having the sequence shown in SEQ ID NO: 1 and described inU.S. Pat. Publ. No. US2010/0216708.

A “cancer cell” or a “tumor cell” as used herein refers to a cancerous,pre-cancerous or transformed cell, either in vivo, ex vivo, and intissue culture, that has spontaneous or induced phenotypic changes thatdo not necessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic nucleic acid, or uptake of exogenousnucleic acid, it can also arise spontaneously or following exposure to acarcinogen, thereby mutating an endogenous gene. Transformation/canceris exemplified by, e.g., morphological changes, immortalization ofcells, aberrant growth control, foci formation, proliferation,malignancy, tumor specific markers levels, invasiveness, tumor growth orsuppression in suitable animal hosts such as nude mice, and the like, invitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manualof Basic Technique (3rd ed. 1994)).

The term “vector” means a polynucleotide capable of being duplicatedwithin a biological system or that can be moved between such systems.Vector polynucleotides typically contain elements, such as origins ofreplication, polyadenylation signal or selection markers that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system. Examples of such biological systems may include acell, virus, animal, plant, and reconstituted biological systemsutilizing biological components capable of duplicating a vector. Thepolynucleotide comprising a vector may be DNA or RNA molecules or ahybrid of these.

The term “expression vector” means a vector that can be utilized in abiological system or in a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

The term “polynucleotide” means a molecule comprising a chain ofnucleotides covalently linked by a sugar-phosphate backbone or otherequivalent covalent chemistry. Double and single-stranded DNAs and RNAsare typical examples of polynucleotides.

The term “polypeptide” or “protein” means a molecule that comprises atleast two amino acid residues linked by a peptide bond to form apolypeptide. Small polypeptides of less than about 50 amino acids may bereferred to as “peptides”.

The term “bispecific EGFR/c-Met molecule” or “bispecific EGFR/c-Met FN3domain containing molecule” as used herein refers to a moleculecomprising an EGFR binding FN3 domain and a distinct c-Met binding FN3domain that are covalently linked together either directly or via alinker. An exemplary bispecific EGFR/c-Met binding molecule comprises afirst FN3 domain specifically binding EGFR and a second FN3 domainspecifically binding c-Met.

“Valent” as used herein refers to the presence of a specified number ofbinding sites specific for an antigen in a molecule. As such, the terms“monovalent”, “bivalent”, “tetravalent”, and “hexavalent” refer to thepresence of one, two, four and six binding sites, respectively, specificfor an antigen in a molecule.

“Mixture” as used herein refers to a sample or preparation of two ormore FN3 domains not covalently linked together. A mixture may consistof two or more identical FN3 domains or distinct FN3 domains.

Compositions of Matter

The present invention provides cysteine engineered monospecific andbispecific EGFR and/or c-Met binding FN3 domain containing molecules andmethods of making and using them.

Monospecific EGFR Binding Molecules

The present invention provides fibronectin type III (FN3) domains thatbind specifically to epidermal growth factor receptor (EGFR) and blockbinding of epidermal growth factor (EGF) to EGFR, and thus can be widelyused in therapeutic and diagnostic applications. The present inventionprovides polynucleotides encoding the FN3 domains of the invention orcomplementary nucleic acids thereof, vectors, host cells, and methods ofmaking and using them.

The FN3 domains of the invention bind EGFR with high affinity andinhibit EGFR signaling, and may provide a benefit in terms ofspecificity and reduced off-target toxicity when compared to smallmolecule EGFR inhibitors, and improved tissue penetration when comparedto conventional antibody therapeutics.

One embodiment of the invention an isolated fibronectin type III (FN3)domain that specifically binds epidermal growth factor receptor (EGFR)and blocks binding of epidermal growth factor (EGF) to EGFR.

The FN3 domains of the invention may block EGF binding to the EGFR withan IC₅₀ value of less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M,less than about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about1×10⁻¹¹ M, or less than about 1×10⁻¹² M in a competition assay employingA431 cells and detecting amount of fluorescence from bound biotinylatedEGF using streptavidin-phycoerythrin conjugate at 600 nM on A431 cellsincubated with or without the FN3 domains of the invention. ExemplaryFN3 domains may block EGF binding to the EGFR with an IC₅₀ value betweenabout 1×10⁻⁹ M to about 1×10⁻⁷ M, such as EGFR binding FN3 domainshaving the amino acid sequence of SEQ ID NOs: 18-29, 107-110, or122-137. The FN3 domains of the invention may block EGF binding to theEGFR by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%/0, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%/0 or 100%when compared to binding of EGF to the EGFR in the absence of the FN3domains of the invention using the same assay conditions.

The FN3 domain of the invention may inhibit EGFR signaling by at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when compared to thelevel of signaling in the absence of FN3 domains of the invention usingthe same assay conditions.

Binding of a ligand such as EGF to EGFR stimulates receptordimerization, autophosphorylation, activation of the receptor'sinternal, cytoplasmic tyrosine kinase domain, and initiation of multiplesignal transduction and transactivation pathways involved in regulationof DNA synthesis (gene activation) and cell cycle progression ordivision. Inhibition of EGFR signaling may result in inhibition in oneor more EGFR downstream signaling pathways and therefore neutralizingEGFR may have various effects, including inhibition of cellproliferation and differentiation, angiogenesis, cell motility andmetastasis.

EGFR signaling may be measured using various well know methods, forexample measuring the autophosphorylation of the receptor at any of thetyrosines Y1068, Y1148, and Y1173 (Downward et al., Nature 311:483-5,1984) and/or phosphorylation of natural or synthetic substrates.Phosphorylation can be detected using well known methods such as anELISA assay or a western blot using a phosphotyrosine specific antibody.Exemplary assays can be found in Panek et al., J Pharmacol Exp Thera283:1433-44, 1997 and Batley et al., Life Sci 62:143-50, 1998.

In one embodiment, the FN3 domain of the invention inhibits EGF-inducedEGFR phosphorylation at EGFR residue position Tyrosine 1173 with an IC₅₀value of less than about 2.5×10⁻⁶ M, for example less than about 1×10⁻⁶M, less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M, less than about1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about 1×10⁻¹¹ M, or lessthan about 1×10⁻¹² M when measured in A431 cells using 50 ng/mL humanEGF.

In one embodiment, the FN3 domain of the invention inhibits EGF-inducedEGFR phosphorylation at EGFR residue position Tyrosine 1173 with an IC₅₀value between about 1.8×10⁻⁸ M to about 2.5×10⁻⁶ M when measured in A431cells using 50 ng/mL human EGF. Such exemplary FN3 domains are thosehaving the amino acid sequence of SEQ ID NOs: 18-29, 107-110, or122-137.

In one embodiment, the FN3 domain of the invention binds human EGFR witha dissociation constant (K_(D)) of less than about 1×10⁻⁸ M, for exampleless than about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about1×10⁻¹¹ M, less than about 1×10⁻¹² M, or less than about 1×10⁻¹³ M asdetermined by surface plasmon resonance or the Kinexa method, aspracticed by those of skill in the art. In some embodiments, the FN3domain of the invention binds human EGFR with a K_(D) of between about2×10⁻¹⁰ to about 1×10⁻⁸ M. The affinity of a FN3 domain for EGFR can bedetermined experimentally using any suitable method. (See, for example,Berzofsky, et al., “Antibody-Antigen Interactions,” In FundamentalImmunology, Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby,Janis Immunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particular FN3domain-antigen interaction can vary if measured under differentconditions (e.g., osmolarity, pH). Thus, measurements of affinity andother antigen-binding parameters (e.g., K_(D), K_(on), K_(off)) arepreferably made with standardized solutions of protein scaffold andantigen, and a standardized buffer, such as the buffer described herein.

Exemplary FN3 domains of the invention that bind EGFR include FN3domains of SEQ ID NOs: 18-29, 107-110, or 122-137.

In one embodiment, the FN3 domain that specifically binds EGFR comprisesan amino acid sequence at least 87% identical to the amino acid sequenceof SEQ ID NO: 27.

In one embodiment, the FN3 domain that specifically binds EGFR comprises

an FG loop comprising the sequence HNVYKDTNX₉RGL (SEQ ID NO: 179) or thesequence LGSYVFEHDVML (SEQ ID NO: 180), wherein X₉ is M or I; and

a BC loop comprising the sequence X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 181).

wherein

-   -   X₁ is A, T, G or D;    -   X₂ is A, D, Y or W;    -   X₃ is P, D or N;    -   X₄ is L or absent;    -   X₅ is D, H, R, G, Y or W;    -   X₆ is G, D or A;    -   X₇ is A, F, G, H or D; and    -   X₈ is Y, F or L.

The FN3 domains of the invention that specifically bind EGFR and inhibitautophosphorylation of EGFR may comprise as a structural feature an FGloop comprising the sequence HNVYKDTNX₉RGL (SEQ ID NO: 179) or thesequence LGSYVFEHDVML (SEQ ID NO: 180), wherein X₉ is M or I. Such FN3domains may further comprise a BC loop of 8 or 9 amino acids in lengthand defined by the sequence X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 181), andinhibit EGFR autophosphorylation with an IC₅₀ value of less than about2.5×10⁻⁶ M, and with an IC₅₀ value of between about between about1.8×10⁻⁸ M to about 2.5×10⁻⁶ M when measured in A431 cells using 50ng/mL human EGF.

The FN3 domains of the invention that specifically bind EGFR and inhibitautophosphorylation of EGFR further comprise the sequence of

(SEQ ID NO: 182) LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNX₉RGLPLSA EFTT,or the sequence

(SEQ ID NO: 183) LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSYVFEHDVMLPLSA EFTT,wherein

-   -   X₁ is A, T, G or D;    -   X₂ is A, D, Y or W;    -   X₃ is P, D or N;    -   X₄ is L or absent;    -   X₅ is D, H, R, G, Y or W;    -   X₆ is G, D or A;    -   X₇ is A, F, G, H or D;    -   X₈ is Y, F or L; and    -   X₉ is M or I

The EGFR binding FN3 domains can be generated and tested for theirability to inhibit EGFR autophosphorylation using well known methods andmethods described herein.

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds EGFR, wherein the FN3 domain comprises the sequenceshown in SEQ ID NOs: 18-29, 107-110, or 122-137.

In some embodiments, the EGFR binding FN3 domains comprise an initiatormethionine (Met) linked to the N-terminus or a cysteine (Cys) linked toa C-terminus of a particular FN3 domain, for example to facilitateexpression and/or conjugation of half-life extending molecules.

Another embodiment of the invention is an isolated fibronectin type III(FN3) domain that specifically binds EGFR and blocks binding of EGF tothe EGFR, wherein the FN3 domain is isolated from a library designedbased on Tencon sequence of SEQ ID NO: 1.

Monospecific c-Met Binding Molecules

The present invention provides fibronectin type III (FN3) domains thatbind specifically to hepatocyte growth factor receptor (c-Met) and blockbinding of hepatocyte growth factor (HGF) to c-Met, and thus can bewidely used in therapeutic and diagnostic applications. The presentinvention provides polynucleotides encoding the FN3 domains of theinvention or complementary nucleic acids thereof, vectors, host cells,and methods of making and using them.

The FN3 domains of the invention bind c-Met with high affinity andinhibit c-Met signaling, and may provide a benefit in terms ofspecificity and reduced off-target toxicity when compared to smallmolecule c-Met inhibitors, and improved tissue penetration when comparedto conventional antibody therapeutics. The FN3 domains of the inventionare monovalent, therefore preventing unwanted receptor clustering andactivation that may occur with other bivalent molecules.

One embodiment of the invention an isolated fibronectin type III (FN3)domain that specifically binds hepatocyte growth factor receptor (c-Met)and blocks binding of hepatocyte growth factor (HGF) to c-Met.

The FN3 domains of the invention may block HGF binding to c-Met with anIC₅₀ value of about less than about 1×10⁻⁷ M, less than about 1×10⁻⁸ M,less than about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less than about1×10⁻¹¹ M, or less than about 1×10⁻¹² M in an assay detecting inhibitionof binding of biotinylated HGF to c-Met-Fc fusion protein in thepresence of the FN3 domains of the invention. Exemplary FN3 domains myblock HGF binding to the c-Met with an IC₅₀ value between about 2×10⁻¹⁰M to about 6×10⁻⁸. The FN3 domains of the invention may block HGFbinding to c-Met by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%/0, 93%, 94%, 95%, 96%, 97%, 98%, 99%/0or 100% when compared to binding of HGF to c-Met in the absence of theFN3 domains of the invention using the same assay conditions.

The FN3 domain of the invention may inhibit c-Met signaling by at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% when compared to thelevel of signaling in the absence of FN3 domains of the invention usingthe same assay conditions.

Binding of HGF to c-Met stimulates receptor dimerization,autophosphorylation, activation of the receptor's internal, cytoplasmictyrosine kinase domain, and initiation of multiple signal transductionand transactivation pathways involved in regulation of DNA synthesis(gene activation) and cell cycle progression or division. Inhibition ofc-Met signaling may result in inhibition in one or more c-Met downstreamsignaling pathways and therefore neutralizing c-Met may have variouseffects, including inhibition of cell proliferation and differentiation,angiogenesis, cell motility and metastasis.

c-Met signaling may be measured using various well know methods, forexample measuring the autophosphorylation of the receptor on at leastone tyrosine residues Y1230, Y1234 or 11235, and/or phosphorylation ofnatural or synthetic substrates.

Phosphorylation can be detected, for example, using an antibody specificfor phosphotyrosine in an ELISA assay or on a western blot. Some assaysfor tyrosine kinase activity (Panek et al., J Pharmacol Exp Thera283:1433-44, 1997; Batley et al., Life Sci 62:143-50, 1998).

In one embodiment, the FN3 domain of the invention inhibits HGF-inducedc-Met phosphorylation at c-Met residue position 1349 with an IC₅₀ valueof less than about 1×10⁻⁶ M, less than about 1×10⁻⁷ M, less than about1×10⁻⁸ M, less than about 1×10⁻⁹ M, less than about 1×10⁻¹⁰ M, less thanabout 1×10⁻¹¹ M, or less than about 1×10⁻¹² M when measured in NCI-H441cells using 100 ng/mL recombinant human HGF.

In one embodiment, the FN3 domain of the invention inhibits HGF-inducedc-Met phosphorylation at c-Met tyrosine Y1349 with an IC₅₀ value betweenabout 4×10⁻⁹ M to about 1×10⁻⁶ M when measured in NCI-H441 cells using100 ng/mL recombinant human HGF.

In one embodiment, the FN3 domain of the invention binds human c-Metwith an dissociation constant (K_(D)) of equal to or less than about1×10⁻⁷ M, 1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M, 1×10⁻¹³M, 1×10⁻¹⁴ M, or 1×10⁻¹⁵ M as determined by surface plasmon resonance orthe Kinexa method, as practiced by those of skill in the art. I someembodiments, the FN3 domain of the invention binds human c-Met with aK_(D) of between about 3×10⁻¹⁰ to about 5×10⁻⁸ M. The affinity of a FN3domain for c-Met can be determined experimentally using any suitablemethod. (See, for example, Berzofsky, et al., “Antibody-AntigenInteractions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press:New York, N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman andCompany: New York, N.Y. (1992); and methods described herein). Themeasured affinity of a particular FN3 domain-antigen interaction canvary if measured under different conditions (e.g., osmolarity, pH).Thus, measurements of affinity and other antigen-binding parameters(e.g., K_(D), K_(on), K_(off)) are preferably made with standardizedsolutions of protein scaffold and antigen, and a standardized buffer,such as the buffer described herein.

Exemplary FN3 domains of the invention that bind c-Met include FN3domains having the amino acid sequence of SEQ ID NOs: 32-49 or 111-114.

In one embodiment, the FN3 domain that specifically binds c-Metcomprises an amino acid sequence at least 83% identical to the aminoacid sequence of SEQ ID NO: 41.

In one embodiment, the FN3 domain that specifically binds c-Metcomprises

-   -   a C strand and a CD loop comprising the sequence DSFX₁₀IRYX₁₁E        X₁₂X₁₃X₁₄X₁₅GX₁₆ (SEQ ID NO: 184), wherein        -   X₁₀ is W, F or V;        -   X₁₁ is D, F or L;        -   X₁₂ is V, F or L;        -   X₁₃ is V, L or T;        -   X₁₄ is V, R, G, L, T or S;        -   X₁₅ is G, S, A, T or K; and        -   X₁₆ is E or D; and    -   a F strand and a FG loop comprising the sequence        TEYX₁₇VX₁₈IX₁₉X₂₀V KGGX₂₁X₂₂SX₂₃ (SEQ ID NO: 185), wherein        -   X₁₇ is Y, W, I, V, G or A;        -   X₁₈ is N, T, Q or G;        -   X₁₉ is L, M, N or I;        -   X₂₀ is G or S;        -   X₂₁ is S, L, G, Y, T, R, H or K;        -   X₂₂ is I, V or L; and        -   X₂₃ is V, T, H, I, P, Y, T or L.

The FN3 domains of the invention that specifically bind c-Met andinhibit autophosphorylation of c-Met further comprises the sequence:

(SEQ ID NO: 186) LPAPKNLVVSRVTEDSARLSWTAPDAAF DSFX₁₀IRYX₁₁EX₁₂X₁₃X₁₄X₁₅GX₁₆AIVLTVPGSERSYDLTGLKPGTEYX₁₇VX₁₈IX₁₉X₂₀VKGG X₂₁X₂₂SX₂₃PLSAEFTT,wherein

-   -   X₁₀ is W, F or V; and    -   X₁₁ is D, F or L;    -   X₁₂ is V, F or L;    -   X₁₃ is V, L or T;    -   X₁₄ is V, R, G, L, T or S;    -   X₁₅ is G, S, A, T or K;    -   X₁₆ is E or D;    -   X₁₇ is Y, W, I, V, G or A;    -   X₁₈ is N, T, Q or G;    -   X₁₉ is L, M, N or I;    -   X₂₀ is G or S;    -   X₂₁ is S, L, G, Y, T, R, H or K;    -   X₂₂ is I, V or L; and    -   X₂₃ is V, T, H, I, P, Y, T or L.

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds c-Met, wherein the FN3 domain comprises the sequenceshown in SEQ ID NOs: 32-49 or 111-114.

Another embodiment of the invention is an isolated fibronectin type III(FN3) domain that specifically binds c-Met and blocks binding of HGF tothe c-Met, wherein the FN3 domain is isolated from a library designedbased on Tencon sequence of SEQ ID NO: 1.

Isolation of EGFR or c-Met FN3 Domains from a Library Based on TenconSequence

Tencon (SEQ ID NO: 1) is a non-naturally occurring fibronectin type III(FN3) domain designed from a consensus sequence of fifteen FN3 domainsfrom human tenascin-C (Jacobs et al., Protein Engineering, Design, andSelection, 25:107-117, 2012; U.S. Pat. Publ. No. 2010/0216708). Thecrystal structure of Tencon shows six surface-exposed loops that connectseven beta-strands as is characteristic to the FN3 domains, thebeta-strands referred to as A, B, C, D, E, F, and G, and the loopsreferred to as AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle,Proc Natl Acad Sci USA 89:8990-8992, 1992; U.S. Pat. No. 6,673,901).These loops, or selected residues within each loop, can be randomized inorder to construct libraries of fibronectin type III (FN3) domains thatcan be used to select novel molecules that bind EGFR. Table 1 showspositions and sequences of each loop and beta-strand in Tencon (SEQ IDNO: 1).

Library designed based on Tencon sequence may thus have randomized FGloop, or randomized BC and FG loops, such as libraries TCL1 or TCL2 asdescribed below. The Tencon BC loop is 7 amino acids long, thus 1, 2, 3,4, 5, 6 or 7 amino acids may be randomized in the library diversified atthe BC loop and designed based on Tencon sequence. The Tencon FG loop is7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may berandomized in the library diversified at the FG loop and designed basedon Tencon sequence. Further diversity at loops in the Tencon librariesmay be achieved by insertion and/or deletions of residues at loops. Forexample, the FG and/or BC loops may be extended by 1-22 amino acids, ordecreased by 1-3 amino acids. The FG loop in Tencon is 7 amino acidslong, whereas the corresponding loop in antibody heavy chains rangesfrom 4-28 residues. To provide maximum diversity, the FG loop may bediversified in sequence as well as in length to correspond to theantibody CDR3 length range of 4-28 residues. For example, the FG loopcan further be diversified in length by extending the loop by additional1, 2, 3, 4 or 5 amino acids.

Library designed based on Tencon sequence may also have randomizedalternative surfaces that form on a side of the FN3 domain and comprisetwo or more beta strands, and at least one loop. One such alternativesurface is formed by amino acids in the C and the F beta-strands and theCD and the FG loops (a C-CD-F-FG surface). A library design based onTencon alternative C-CD-F-FG surface and is shown in FIG. 4 and detailedgeneration of such libraries is described in U.S. patent applicationSer. No. 13/852,930.

Library designed based on Tencon sequence also includes librariesdesigned based on Tencon variants, such as Tencon variants havingsubstitutions at residues positions 11, 14, 17, 37, 46, 73, or 86(residue numbering corresponding to SEQ ID NO: 1), and which variantsdisplay improve thermal stability. Exemplary Tencon variants aredescribed in US Pat. Publ. No. 2011/0274623, and include Tencon27 (SEQID NO: 99) having substitutions E11R, L17A, N46V, E86I when compared toTencon of SEQ ID NO: 1.

TABLE 1 Tencon FN3 domain (SEQ ID NO: 1) A strand  1-12 AB loop 13-16 Bstrand 17-21 BC loop 22-28 C strand 29-37 CD loop 38-43 D strand 44-50DE loop 51-54 E strand 55-59 EF loop 60-64 F strand 65-74 FG loop 75-81G strand 82-89

Tencon and other FN3 sequence based libraries can be randomized atchosen residue positions using a random or defined set of amino acids.For example, variants in the library having random substitutions can begenerated using NNK codons, which encode all 20 naturally occurringamino acids. In other diversification schemes, DVK codons can be used toencode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp,Glu, Gly, and Cys. Alternatively, NNS codons can be used to give rise toall 20 amino acid residues and simultaneously reducing the frequency ofstop codons. Libraries of FN3 domains with biased amino aciddistribution at positions to be diversified can be synthesized forexample using Slonomics® technology (http:_//www_sloning_com). Thistechnology uses a library of pre-made double stranded triplets that actas universal building blocks sufficient for thousands of gene synthesisprocesses. The triplet library represents all possible sequencecombinations necessary to build any desired DNA molecule. The codondesignations are according to the well known IUB code.

The FN3 domains specifically binding EGFR or c-Met of the invention canbe isolated by producing the FN3 library such as the Tencon libraryusing cis display to ligate DNA fragments encoding the scaffold proteinsto a DNA fragment encoding RepA to generate a pool of protein-DNAcomplexes formed after in vitro translation wherein each protein isstably associated with the DNA that encodes it (U.S. Pat. No. 7,842,476;Odegrip et al., Proc Natl Acad Sci USA 101, 2806-2810, 2004), andassaying the library for specific binding to EGFR and/or c-Met by anymethod known in the art and described in the Example. Exemplary wellknown methods which can be used are ELISA, sandwich immunoassays, andcompetitive and non-competitive assays (see, e.g., Ausubel et al., eds,1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons,Inc., New York). The identified FN3 domains specifically binding EGFR orc-Met are further characterized for their ability to block EGFR ligandsuch as EGF binding to EGFR, or HGF binding to c-Met, and for theirability to inhibit EGFR and/or c-Met signaling using methods describedherein.

The FN3 domains specifically binding to EGFR or c-Met of the inventioncan be generated using any FN3 domain as a template to generate alibrary and screening the library for molecules specifically bindingEGFR or c-Met using methods provided within. Exemplar FN3 domains thatcan be used are the 3rd FN3 domain of tenascin C (TN3) (SEQ ID NO: 75),Fibcon (SEQ ID NO: 76), and the 10^(th) FN3 domain of fibronectin (FN10)(SEQ ID NO: 77). Standard cloning and expression techniques are used toclone the libraries into a vector or synthesize double stranded cDNAcassettes of the library, to express, or to translate the libraries invitro. For example ribosome display (Hanes and Pluckthun, Proc Natl AcadSci USA, 94, 4937-4942, 1997), mRNA display (Roberts and Szostak, ProcNatl Acad Sci USA, 94, 12297-12302, 1997), or other cell-free systems(U.S. Pat. No. 5,643,768) can be used. The libraries of the FN3 domainvariants may be expressed as fusion proteins displayed on the surfacefor example of any suitable bacteriophage. Methods for displaying fusionpolypeptides on the surface of a bacteriophage are well known (U.S. Pat.Publ. No. 2011/0118144; Int. Pat. Publ. No. WO2009/085462; U.S. Pat.Nos. 6,969,108; 6,172,197; 5,223,409; 6,582,915; 6,472,147).

The FN3 domains specifically binding EGFR or c-Met of the invention canbe modified to improve their properties such as improve thermalstability and reversibility of thermal folding and unfolding. Severalmethods have been applied to increase the apparent thermal stability ofproteins and enzymes, including rational design based on comparison tohighly similar thermostable sequences, design of stabilizing disulfidebridges, mutations to increase alpha-helix propensity, engineering ofsalt bridges, alteration of the surface charge of the protein, directedevolution, and composition of consensus sequences (Lehmann and Wyss,Curr Opin Biotechnol, 12, 371-375, 2001). High thermal stability mayincrease the yield of the expressed protein, improve solubility oractivity, decrease immunogenicity, and minimize the need of a cold chainin manufacturing. Residues that can be substituted to improve thermalstability of Tencon (SEQ ID NO: 1) are residue positions 11, 14, 17, 37,46, 73, or 86, and are described in US Pat. Publ. No. 2011/0274623.Substitutions corresponding to these residues can be incorporated to theFN3 domains or the bispecific FN3 domain containing molecules of theinvention.

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds EGFR and blocks binding of EGF to EGFR, comprisingthe sequence shown in SEQ ID NOs: 18-29, 107-110, 122-137, furthercomprising substitutions at one or more residue positions correspondingto positions 11, 14, 17, 37, 46, 73, and 86 in Tencon (SEQ ID NO: 1).

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds c-Met and blocks binding of HGF to c-Met, comprisingthe sequence shown in SEQ ID NOs: 32-49 or 111-114, further comprisingsubstitutions at one or more residue positions corresponding topositions 11, 14, 17, 37, 46, 73, and 86 in Tencon (SEQ ID NO: 1).

Exemplary substitutions are substitutions E11N, E14P, L17A, E37P, N46V,G73Y and E86I (numbering according to SEQ ID NO: 1).

In some embodiments, the FN3 domains of the invention comprisesubstitutions corresponding to substitutions L17A, N46V, and E86I inTencon (SEQ ID NO: 1).

The FN3 domains specifically binding EGFR (FIG. 1) have an extended FGloop when compared to Tencon (SEQ ID NO: 1). Therefore, the residuescorresponding to residues 11, 14, 17, 37, 46, 73, and 86 in Tencon (SEQID NO: 1) are residues 11, 14, 17, 37, 46, 73 and 91 in EGFR FN3 domainsshown in FIGS. 1A and 1B except for the FN3 domain of SEQ ID NO: 24,wherein the corresponding residues are residues 11, 14, 17, 38, 74, and92 due to an insertion of one amino acid in the BC loop.

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds EGFR and blocks binding of EGF to EGFR comprising theamino acid sequence shown in SEQ ID NOs: 18-29, 107-110, or 122-137,optionally having substitutions corresponding to substitutions L17A,N46V, and E86I in Tencon (SEQ ID NO: 1).

Another embodiment of the invention is an isolated FN3 domain thatspecifically binds c-Met and blocks binding of HGF to c-Met comprisingthe amino acid sequence shown in SEQ ID NOs: 32-49 or 111-114,optionally having substitutions corresponding to substitutions L17A,N46V, and E86I in Tencon (SEQ ID NO: 1).

Measurement of protein stability and protein lability can be viewed asthe same or different aspects of protein integrity. Proteins aresensitive or “labile” to denaturation caused by heat, by ultraviolet orionizing radiation, changes in the ambient osmolarity and pH if inliquid solution, mechanical shear force imposed by small pore-sizefiltration, ultraviolet radiation, ionizing radiation, such as by gammairradiation, chemical or heat dehydration, or any other action or forcethat may cause protein structure disruption. The stability of themolecule can be determined using standard methods. For example, thestability of a molecule can be determined by measuring the thermalmelting (“TM”) temperature, the temperature in ° Celsius (° C.) at whichhalf of the molecules become unfolded, using standard methods.Typically, the higher the TM, the more stable the molecule. In additionto heat, the chemical environment also changes the ability of theprotein to maintain a particular three dimensional structure.

In one embodiment, the FN3 domains binding EGFR or c-Met of theinvention exhibit increased stability by at least 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%/0, 85%, 90%,or 95% or more compared to the same domain prior to engineering measuredby the increase in the TM.

Chemical denaturation can likewise be measured by a variety of methods.Chemical denaturants include guanidinium hydrochloride, guanidiniumthiocyanate, urea, acetone, organic solvents (DMF, benzene,acetonitrile), salts (ammonium sulfate lithium bromide, lithiumchloride, sodium bromide, calcium chloride, sodium chloride); reducingagents (e.g. dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene,and hydrides, such as sodium borohydride), non-ionic and ionicdetergents, acids (e.g. hydrochloric acid (HCl), acetic acid (CH₃COOH),halogenated acetic acids), hydrophobic molecules (e.g. phosopholipids),and targeted denaturants. Quantitation of the extent of denaturation canrely on loss of a functional property, such as ability to bind a targetmolecule, or by physiochemical properties, such as tendency toaggregation, exposure of formerly solvent inaccessible residues, ordisruption or formation of disulfide bonds.

In one embodiment, the FN3 domains of the invention binding EGFR orc-Met exhibit increased stability by at least 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%/0, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%/0, 85%, 90%, or95% or more compared to the same scaffold prior to engineering measuredby using guanidinium hydrochloride as a chemical denaturant. Increasedstability can be measured as a function of decreased tryptophanfluorescence upon treatment with increasing concentrations of guanidinehydrochloride using well known methods.

The FN3 domains of the invention may be generated as monomers, dimers,or multimers, for example, as a means to increase the valency and thusthe avidity of target molecule binding, or to generate bi- ormultispecific scaffolds simultaneously binding two or more differenttarget molecules. The dimers and multimers may be generated by linkingmonospecific, bi- or multispecific protein scaffolds, for example, bythe inclusion of an amino acid linker, for example a linker containingpoly-glycine, glycine and serine, or alanine and proline. Exemplarylinker include (GS)₂, (SEQ ID NO: 78), (GGGGS)₅ (SEQ ID NO: 79), (AP)₂(SEQ ID NO: 80), (AP)₅ (SEQ ID NO: 81), (AP)₁₀ (SEQ ID NO: 82), (AP)₂₀(SEQ ID NO: 83), A(EAAAK)₅AAA (SEQ ID NO: 84), linkers. The dimers andmultimers may be linked to each other in a N- to C-direction. The use ofnaturally occurring as well as artificial peptide linkers to connectpolypeptides into novel linked fusion polypeptides is well known in theliterature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthanet al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer, Biochemistry35, 109-116, 1996; U.S. Pat. No. 5,856,456).

Bispecific EGFR/c/Met Binding Molecules

The bispecific EGFR/c-Met FN3 domain containing molecules of theinvention may provide a benefit in terms of specificity and reducedoff-target toxicity when compared to small molecule EGFR inhibitors, andimproved tissue penetration when compared to conventional antibodytherapeutics. The present invention is based at least in part on thesurprising finding that the bispecific EGFR/c-Met FN3 domain containingmolecules of the invention provide a significantly improved synergisticinhibitory effect when compared to a mixture of EGFR-binding andc-Met-binding FN3 domains. The molecules may be tailored to specificaffinity towards both EGFR and c-Met to maximize tumor penetration andretention.

One embodiment of the invention is an isolated bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain, wherein the first FN3 domain specifically bindsepidermal growth factor receptor (EGFR) and blocks binding of epidermalgrowth factor (EGF) to EGFR, and the second FN3 domain specificallybinds hepatocyte growth factor receptor (c-Met), and blocks binding ofhepatocyte growth factor (HGF) to c-Met.

The bispecific EGFR/c-Met FN3 domain containing molecules of theinvention can be generated by covalently linking any EGFR-binding FN3domain and any c-Met-binding FN3 domain of the invention directly or viaa linker. Therefore, the first FN3 domain of the bispecific molecule mayhave characteristics as described above for the EGFR-binding FN3domains, and the second FN3 domain of the bispecific molecule may havecharacteristics as described above for the c-Met-binding FN3 domains.

In one embodiment, the first FN3 domain of the bispecific EGFR/c-Met FN3domain containing molecule inhibits EGF-induced EGFR phosphorylation atEGFR residue Tyrosine 1173 with an IC₅₀ value of less than about2.5×10⁻⁸ M when measured in A431 cells using 50 ng/mL human EGF, and thesecond FN3 domain of the bispecific EGFR/c-Met FN3 domain containingmolecule inhibits HGF-induced c-Met phosphorylation at c-Met residueTyrosine 1349 with an IC₅₀ value of less than about 1.5×10⁻⁶ M whenmeasured in NCI-H441 cells using 100 ng/mL human HGF.

In another embodiment, the first FN3 domain of the bispecific EGFR/c-MetFN3 domain containing molecule inhibits EGF-induced EGFR phosphorylationat EGFR residue Tyrosine 1173 with an IC₅₀ value of between about1.8×10⁻⁸ M to about 2.5×10⁻⁶ M when measured in A431 cells using 50ng/mL human EGF, and the second FN3 domain of the bispecific EGFR/c-MetFN3 domain containing molecule inhibits HGF-induced c-Metphosphorylation at c-Met residue Tyrosine 1349 with an IC₅₀ valuebetween about 4×10⁻⁹ M to about 1.5×10⁻⁶ M when measured in NCI-H441cells using 100 ng/mL human HGF.

In another embodiment, the first FN3 domain of the bispecific EGFR/c-MetFN3 domain containing molecule binds human EGFR with a dissociationconstant (K_(D)) of less than about 1×10⁻⁸ M, and the second FN3 domainof the bispecific EGFR/c-Met FN3 domain containing molecule binds humanc-Met with a K_(D) of less than about 5×10⁻⁸ M.

In the bispecific molecule binding both EGFR and c-Met, the first FN3domain binds human EGFR with a K_(D) of between about 2×10⁻¹⁰ to about1×10⁻⁸ M, and the second FN3 domain binds human c-Met with a K_(D) ofbetween about 3×10⁻¹⁰ to about 5×10⁻⁸ M.

The affinity of the bispecific EGFR/c-Met molecule for EGFR and c-Metcan be determined as described above for the monospecific molecules.

The first FN3 domain in the bispecific EGFR/c-Met molecule of theinvention may block EGF binding to EGFR with an IC₅₀ value of betweenabout 1×10⁻⁹ M to about 1.5×10⁻⁷ M in an assay employing A431 cells anddetecting amount of fluorescence from bound biotinylated EGF usingstreptavidin-phycoerythrin conjugate at 600 nM on A431 cells incubatedwith or without the first FN3 domain. The first FN3 domain in thebispecific EGFR/c-Met molecule of the invention may block EGF binding tothe EGFR by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% whencompared to binding of EGF to EGFR in the absence of the first FN3domains using the same assay conditions.

The second FN3 domain in the bispecific EGFR/c-Met molecule of theinvention may block HGF binding to c-Met with an IC₅₀ value of betweenabout 2×10⁻¹⁰ M to about 6×10⁻⁸ M in an assay detecting inhibition ofbinding of biotinylated HGF to c-Met-Fc fusion protein in the presenceof the second FN3 domain. The second FN3 domain in the bispecificEGFR/c-Met molecule may block HGF binding to c-Met by at least 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 920/0, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% when compared to binding of HGF toc-Met in the absence of the second FN3 domain using the same assayconditions.

The bispecific EGFR/c-Met molecule of the invention may inhibit EGFRand/or c-Met signaling by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% when compared to the level of signaling in the absence ofthe bispecific EGFR/c-Met molecule of the invention using the same assayconditions.

EGFR and c-Met signaling may be measured using various well know methodsas described above for the monospecific molecules.

The bispecific EGFR/c-Met molecules of the invention comprising thefirst FN3 domain specifically binding EGFR and the second FN3 domainspecifically binding c-Met provide a significantly increased synergisticinhibition of EGFR and c/Met signaling and tumor cell proliferation whencompared to the synergistic inhibition observed by a mixture of thefirst and the second FN3 domain. Synergistic inhibition can be assessedfor example by measuring inhibition of ERK phosphorylation by thebispecific EGFR/c-Met FN3 domain containing molecules and by a mixtureof two monospecific molecules, one binding EGFR and the other c-Met. Thebispecific EGFR/c-Met molecules of the invention may inhibit ERKphosphorylation with an IC₅₀ value at least about 100 fold smaller, forexample at least 500, 1000, 5000 or 10,000 fold smaller when compared tothe IC₅₀ value for a mixture of two monospecific FN3 domains, indicatingat least 100 fold increased potency for the bispecific EGFR/c-Met FN3domain containing molecules when compared to the mixture of twomonospecific FN3 domains. Exemplary bispecific EGFR-c-Met FN3 domaincontaining molecules may inhibit ERK phosphorylation with and IC₅₀ valueof about 5×10⁻⁹ M or less. ERK phosphorylation can be measured usingstandard methods and methods described herein.

The bispecific EGFR/c-Met FN3 domain containing molecule of theinvention may inhibit H292 cell proliferation with an IC₅₀ value that isat least 30-fold less when compared to the IC₅₀ value of inhibition ofH292 cell growth with a mixture of the first FN3 domain and the secondFN3, wherein the cell proliferation is induced with medium containing10% FBS supplemented with 7.5 ng/mL HGF. The bispecific molecule of theinvention may inhibit tumor cell proliferation with an IC₅₀ value thatis about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600,700, 800, or about 1000 fold less when compared to the IC₅₀ value ofinhibition of tumor cell proliferation with a mixture of the first FN3domain and the second FN3 domain. Inhibition of tumor cell proliferationcan be measured using standard methods and methods described herein.

Another embodiment of the invention is a bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain, wherein the first FN3 domain specifically bindsepidermal growth factor receptor (EGFR) and blocks binding of epidermalgrowth factor (EGF) to EGFR, and the second FN3 domain specificallybinds hepatocyte growth factor receptor (c-Met), and blocks binding ofhepatocyte growth factor (HGF) to c-Met, wherein

the first FN3 domain comprises

-   -   an FG loop comprising the sequence HNVYKDTNX₉RGL (SEQ ID        NO: 179) or the sequence LGSYVFEHDVML (SEQ ID NO: 180), wherein        X₉ is M or I; and    -   a BC loop comprising the sequence X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO:        181),    -   wherein        -   X₁ is A, T, G or D;        -   X₂ is A, D, Y or W;        -   X₃ is P, D or N;        -   X₄ is L or absent;        -   X₅ is D, H, R, G, Y or W;        -   X₆ is G, D or A;        -   X₇ is A, F, G, H or D; and        -   X₈ is Y, F or L; and

the second FN3 domain comprises

-   -   a C strand and a CD loop comprising the sequence DSFX₁₀IRYX₁₁E        X₁₂X₁₃X₁₄X₁₅GX₁₆ (SEQ ID NO: 184), wherein        -   X₁₀ is W, F or V;        -   X₁₁ is D, F or L;        -   X₁₂ is V, F or L;        -   X₁₃ is V, L or T;        -   X₁₄ is V, R, G, L, T or S;        -   X₁₅ is G, S, A, T or K; and        -   X₁₆ is E or D; and    -   a F strand and a FG loop comprising the sequence        TEYX₁₇VX₁₈IX₁₉X₂₀V KGGX₂₁X₂₂SX₂₃ (SEQ ID NO: 185), wherein        -   X₁₇ is Y, W, I, V, G or A;        -   X₁₈ is N, T, Q or G;        -   X₁₉ is L, M, N or I;        -   X₂₀ is G or S;        -   X₂₁ is S, L, G, Y, T, R, H or K;        -   X₂₂ is I, V or L; and        -   X₂₃ is V, T, H, I, P, Y, T or L.

In another embodiment, the bispecific molecule comprises the first FN3domain that binds EGFR comprising the sequence:

(SEQ ID NO: 182) LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNX₉RGL PLS AEFTT,or the sequence

(SEQ ID NO: 183) LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈ DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV LGSYVFEHDVMLPLSA EFTT,wherein in the SEQ ID NOs: X and X;

-   -   X₁ is A, T, G or D;    -   X₂ is A, D, Y or W;    -   X₃ is P, D or N;    -   X₄ is L or absent;    -   X₅ is D, H, R, G, Y or W;    -   X₆ is G, D or A;    -   X₇ is A, F, G, H or D;    -   X₈ is Y, F or L; and    -   X₉ is M or I.

In another embodiment, the bispecific molecule comprises the second FN3domain that binds c-Met comprising the sequence

(SEQ ID NO: 186) LPAPKNLVVSRVTEDSARLSWTAPDAAF DSFX₁₀IRYX₁₁EX₁₂X₁₃X₁₄X₁₅GX₁₆AIVLTVPGSERSYDLTGLKPG TEYX₁₇VX₁₈IX₁₉X₂₀VKG GX₂₁X₂₂SX₂₃PLSAEFTT,wherein

-   -   X₁₀ is W, F or V; and    -   X₁₁ is D, F or L;    -   X₁₂ is V, F or L;    -   X₁₃ is V, L or T;    -   X₁₄ is V, R, G, L, T or S;    -   X₁₅ is G, S, A, T or K;    -   X₁₆ is E or D;    -   X₁₇ is Y, W, I, V, G or A;    -   X₁₈ is N, T, Q or G;    -   X₁₉ is L, M, N or I;    -   X₂₀ is G or S;    -   X₂₁ is S, L, G, Y, T, R, H or K;    -   X₂₂ is I, V or L; and    -   X₂₃ is V, T, H, I, P, Y, T or L.

Exemplary bispecific EGFR/c-Met FN3 domain containing molecules comprisethe amino acid sequence shown in SEQ ID NOs: 50-72, 106, 118-121, or138-165.

The bispecific EGFR/c-Met molecules of the invention comprise certainstructural characteristics associated with their functionalcharacteristics, such as inhibition of EGFR autophosphorylation, such asthe FG loop of the first FN3 domain that binds EGFR comprising thesequence HNVYKDTNX₉RGL (SEQ ID NO: 179) or the sequence LGSYVFEHDVML(SEQ ID NO: 180), wherein X₉ is M or I.

In one embodiment, the bispecific EGFR/c-Met FN3 domain containingmolecules of the invention

-   -   inhibit EGF-induced EGFR phosphorylation at EGFR residues        Tyrosine 1173 with and IC₅₀ value of less than about 8×10⁻⁷ M        when measured in A431 cells using 50 ng/mL human EGF;    -   inhibit HGF-induced c-Met phosphorylation at c-Met residues        Tyrosine 1349 with and IC₅₀ value of less than about 8.4×10⁻⁷ M        when measured in NCI-H441 cells using 100 ng/mL human HGF;    -   inhibit HGF-induced NCI-H292 cell proliferation with an IC₅₀        value of less than about 9.5×10⁻⁶ M wherein the cell        proliferation is induced with 10% FBS containing 7.5 ng HGF;    -   bind EGFR with a K_(D) of less than about 2.0×10⁻⁸ M;    -   bind c-Met with a K_(D) of less than about 2.0×10⁻⁸ M.

In another embodiment, the bispecific EGFR/c-Met FN3 domain containingmolecules of the invention

-   -   inhibit EGF-induced EGFR phosphorylation at EGFR residues        Tyrosine 1173 with and IC₅₀ of between about 4.2×10⁻⁹ M and        8×10⁻⁷ M when measured in A431 cells using 50 ng/mL human EGF;    -   inhibit HGF-induced c-Met phosphorylation at c-Met residues        Tyrosine 1349 with and IC₅₀ value of between about 2.4×10⁻⁸ M to        about 8.4×10⁻⁷ M when measured in NCI-H441 cells using 100 ng/mL        human HGF;    -   inhibit HGF-induced NCI-H292 cell proliferation with an IC₅₀        value between about 2.3×10⁻⁸ M to about 9.5×10⁻⁶ M wherein the        cell proliferation is induced with 10% FBS containing 7.5 ng        HGF;    -   bind EGFR with a K_(D) of between about 2×10⁻¹⁰ M to about        2.0×10 M;    -   bind c-Met with a K_(D) of between about 1×10⁻⁹ M to about        2.0×10⁻⁸ M.

In one embodiment, bispecific EGFR/c-Met molecules comprise theEGFR-binding FN3 domain comprising the sequence

(SEQ ID NO: 182) LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV HNVYKDTNX₉RGL PL SAEFTT,wherein

-   -   X₁ is D;    -   X₂ is D;    -   X₃ is P;    -   X₄ is absent;    -   X₅ is H or W;    -   X₆ is A;    -   X₇ is F    -   X₈ is Y; and    -   X₉ is M or I; and

the c-Met-binding FN3 domain comprising the sequence

(SEQ ID NO: 186) PAPKNLVVSRVTEDSARLSWTAPDAAF DSFX₁₀IRYX₁₁EX₁₂X₁₃X₁₄X₁₅GX₁₆AIVLTVPGSERSYDLTGLKPG TEYX₁₇VX₁₈IX₁₉X₂₀VKG GX₂₁X₂₂SX₂₃ PLSAEFTT,wherein

-   -   X₁₀ is W;    -   X₁₁ is F;    -   X₁₂ is F;    -   X₁₃ is V or L;    -   X₁₄ is G or S;    -   X₁₅ is S or K;    -   X₁₆ is E or D;    -   X₁₇ is V;    -   X₁₈ is N;    -   X₁₉ is L or M;    -   X₂₀ is G or S;    -   X₂₁ is S or K;    -   X₂₂ is I; and    -   X₂₃ is P.

Exemplary bispecific EGFR/c-Met molecules are those having the sequenceshown in SEQ ID NOs: 57, 61, 62, 63, 64, 65, 66, 67 and 68.

The bispecific molecules of the invention may further comprisesubstitutions at one or more residue positions in the first FN3 domainand/or the second FN3 domain corresponding to positions 11, 14, 17, 37,46, 73, and 86 in Tencon (SEQ ID NO: 1) as described above, and asubstitution at position 29. Exemplary substitutions are substitutionsE11N, E14P, L17A, E37P, N46V, G73Y, E86I and D29E (numbering accordingto SEQ ID NO: 1). Skilled in the art will appreciate that other aminoacids can be used for substitutions, such as amino acids within a familyof amino acids that are related in their side chains as described infra.The generated variants can be tested for their stability and binding toEGFR and/or c-Met using methods herein.

In one embodiment, the bispecific EGFR/c-Met FN3 domain containingmolecule comprises the first FN3 domain that binds specifically EGFR andthe second FN3 domain that binds specifically c-Met, wherein the firstFN3 domain comprises the sequence:

(SEQ ID NO: 187) LPAPKNLVVSX₂₄VTX₂₅DSX₂₆RLSWDDPX₂₇AFYX₂₈SFLIQYQX₂₉SEKVGEAIX₃₀LTVPGSERSYDLTGLKPGTEYTVSIYX₃₁VHNVYKDTN X₃₂RGLPLSAX₃₃FTT,whereinX₂₄ is E, N or R;X₂₅ is E or P;X₂₆ is L or A;X₂₇ is H or W;X₂₈ is E or D;X₂₉ is E or P;X₃₀ is N or V;X₃₁ is G or Y;X₃₂ is M or I; andX₃₃ is E or I;

and the second FN3 domain comprises the sequence:

(SEQ ID NO: 188) LPAPKNLVVSX₃₄VTX₃₅DSX₃₆RLSWTAPDAAFDSFWIRYFX₃₇F₃₈X₃₉X₄₀GX₄₁AIX₄₂LTVPGSERSYDLTGLKPGTEYVVNIX₄₃X₄₄VKGG X₄₅ISPPLSAX₄₆FTT;whereinX₃₄ is E, N or R;X₃₅ is E or P;X₃₆ is L or A;X₃₇ is E or P;X₃₈ is V or L;X₃₉ is G or S;X₄₀ is S or K;X₄₁ is E or D;X₄₂ is N or V;X₄₃ is L or M;X₄₄ is G or S;X₄₅ is S or K; andX₄₆ is E or I.

In other embodiments, the bispecific EGFR/c-Met FN3 domain containingmolecule comprises the first FN3 domain comprising an amino acidsequence at least 87% identical to the amino acid sequence of SEQ ID NO:27, and the second FN3 domain comprising an amino acid sequence at least83% identical to the amino acid sequence of SEQ ID NO: 41.

The bispecific EGFR/c-Met FN3 domain containing molecules of theinvention may be tailored to a specific affinity towards EGFR and c-Metto maximize tumor accumulation.

Another embodiment of the invention is an isolated bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain, wherein the first FN3 domain specifically bindsepidermal growth factor receptor (EGFR) and blocks binding of epidermalgrowth factor (EGF) to EGFR, and the second FN3 domain specificallybinds hepatocyte growth factor receptor (c-Met), and blocks binding ofhepatocyte growth factor (HGF) to c-Met, wherein the first FN3 domainand the second FN3 domain is isolated from a library designed based onTencon sequence of SEQ ID NO: 1.

The bispecific EGFR/c-Met FN3 domain containing molecule of theinvention can be generated by covalently coupling the EGFR-binding FN3domain and the c-Met binding FN3 domain of the invention using wellknown methods. The FN3 domains may be linked via a linker, for example alinker containing poly-glycine, glycine and serine, or alanine andproline. Exemplary linker include (GS)₂, (SEQ ID NO: 78), (GGGGS)₅ (SEQID NO: 79), (AP)₂ (SEQ ID NO: 80), (AP)₅ (SEQ ID NO: 81), (AP)₁₀ (SEQ IDNO: 82), (AP)₂₀ (SEQ ID NO: 83), A(EAAAK)₅AAA (SEQ ID NO: 84), linkers.The use of naturally occurring as well as artificial peptide linkers toconnect polypeptides into novel linked fusion polypeptides is well knownin the literature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989;Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer,Biochemistry 35, 109-116, 1996; U.S. Pat. No. 5,856,456). The bispecificEGFR/c-Met molecules of the invention may be linked together from aC-terminus of the first FN3 domain to the N-terminus of the second FN3domain, or from the C-terminus of the second FN3 domain to theN-terminus of the first FN3 domain. Any EGFR-binding FN3 domain may becovalently linked to a c-Met-binding FN3 domain. Exemplary EGFR-bindingFN3 domains are domains having the amino acid sequence shown in SEQ IDNOs: 18-29, 107-110, and 122-137, and exemplary c-Met binding FN3domains are domains having the amino acid sequence shown in SEQ ID NOs:32-49 and 111-114. The EGFR-binding FN3 domains to be coupled to abispecific molecule may additionally comprise an initiator methionine(Met) at their N-terminus.

Variants of the bispecific EGFR/c-Met FN3 domain containing moleculesare within the scope of the invention. For example, substitutions can bemade in the bispecific EGFR/c-Met FN3 domain containing molecule as longas the resulting variant retains similar selectivity and potency towardsEGFR and c-Met when compared to the parent molecule. Exemplarymodifications are for example conservative substitutions that willresult in variants with similar characteristics to those of the parentmolecules. Conservative replacements are those that take place within afamily of amino acids that are related in their side chains. Geneticallyencoded amino acids can be divided into four families: (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine); (3)nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. Alternatively, the amino acid repertoire can be grouped as(1) acidic (aspartate, glutamate); (2) basic (lysine, argininehistidine), (3) aliphatic (glycine, alanine, valine, leucine,isoleucine, serine, threonine), with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (Stryer (ed.), Biochemistry,2nd ed, WH Freeman and Co., 1981). Non-conservative substitutions can bemade to the bispecific EGFR/c-Met FN3 domain containing molecule thatinvolves substitutions of amino acid residues between different classesof amino acids to improve properties of the bispecific molecules.Whether a change in the amino acid sequence of a polypeptide or fragmentthereof results in a functional homolog can be readily determined byassessing the ability of the modified polypeptide or fragment to producea response in a fashion similar to the unmodified polypeptide orfragment using the assays described herein. Peptides, polypeptides orproteins in which more than one replacement has taken place can readilybe tested in the same manner.

The bispecific EGFR/c-Met FN3 domain containing molecules of theinvention may be generated as dimers or multimers, for example, as ameans to increase the valency and thus the avidity of target moleculebinding. The multimers may be generated by linking one or moreEGFR-binding FN3 domains and one or more c-Met-binding FN3 domain toform molecules comprising at least three individual FN3 domains that areat least bispecific for either EGFR or c-Met, for example by theinclusion of an amino acid linker using well known methods.

Another embodiment of the invention is a bispecific FN3 domaincontaining molecule comprising a first fibronectin type III (FN3) domainand a second FN3 domain, wherein the first FN3 domain specifically bindsepidermal growth factor receptor (EGFR) and blocks binding of epidermalgrowth factor (EGF) to EGFR, and the second FN3 domain specificallybinds hepatocyte growth factor receptor (c-Met), and blocks binding ofhepatocyte growth factor (HGF) to c-Met comprising the amino acidsequence shown in SEQ ID NOs: 50-72 or 106.

Half-Life Extending Moieties

The bispecific EGFR/c-Met FN3 domain containing molecules or themonospecific EGFR or c-Met binding FN3 domains of the present inventionmay incorporate other subunits for example via covalent interaction. Inone aspect of the invention, the bispecific EGFR/c-Met FN3 domaincontaining molecules of the invention further comprise a half-lifeextending moiety. Exemplary half-life extending moieties are albumin,albumin-binding proteins and/or domains, transferrin and fragments andanalogues thereof, and Fc regions. An exemplary albumin-binding domainis shown in SEQ ID NO: 117.

All or a portion of an antibody constant region may be attached to themolecules of the invention to impart antibody-like properties,especially those properties associated with the Fc region, such as Fceffector functions such as C1q binding, complement dependentcytotoxicity (CDC), Fc receptor binding, antibody-dependentcell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cellsurface receptors (e.g., B cell receptor, BCR), and can be furthermodified by modifying residues in the Fc responsible for theseactivities (for review; see Strohl, Curr Opin Biotechnol. 20, 685-691,2009).

Additional moieties may be incorporated into the bispecific molecules ofthe invention such as polyethylene glycol (PEG) molecules, such asPEG5000 or PEG20,000, fatty acids and fatty acid esters of differentchain lengths, for example laurate, myristate, stearate, arachidate,behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid,octadecanedioic acid, docosanedioic acid, and the like, polylysine,octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides)for desired properties. These moieties may be direct fusions with theprotein scaffold coding sequences and may be generated by standardcloning and expression techniques. Alternatively, well known chemicalcoupling methods may be used to attach the moieties to recombinantlyproduced molecules of the invention.

A pegyl moiety may for example be added to the bispecific ormonospecific molecules of the invention by incorporating a cysteineresidue to the C-terminus of the molecule and attaching a pegyl group tothe cysteine using well known methods. Exemplary bispecific moleculeswith the C-terminal cysteine are those having the amino acid sequenceshown in SEQ IN NO: 170-178.

Monospecific and bispecific molecules of the invention incorporatingadditional moieties may be compared for functionality by several wellknown assays. For example, altered properties of monospecific and/orbispecific molecules due to incorporation of Fc domains and/or Fc domainvariants may be assayed in Fc receptor binding assays using solubleforms of the receptors, such as the FcγRI, FcγRII, FcγRIII or FcRnreceptors, or using well known cell-based assays measuring for exampleADCC or CDC, or evaluating pharmacokinetic properties of the moleculesof the invention in in vivo models.

Polynucleotides, Vectors, Host Cells

The invention provides for nucleic acids encoding the EGFR-binding orc-Met binding FN3 domains or the bispecific EGFR/c-Met FN3 domaincontaining molecules of the invention as isolated polynucleotides or asportions of expression vectors or as portions of linear DNA sequences,including linear DNA sequences used for in vitrotranscription/translation, vectors compatible with prokaryotic,eukaryotic or filamentous phage expression, secretion and/or display ofthe compositions or directed mutagens thereof. Certain exemplarypolynucleotides are disclosed herein, however, other polynucleotideswhich, given the degeneracy of the genetic code or codon preferences ina given expression system, encode the protein scaffolds and libraries ofthe protein scaffolds of the invention are also within the scope of theinvention.

One embodiment of the invention is an isolated polynucleotide encodingthe FN3 domain specifically binding EGFR having the amino acid sequenceof SEQ ID NOs: 18-29, 107-110, or 122-137.

One embodiment of the invention is an isolated polynucleotide comprisingthe polynucleotide sequence of SEQ ID NOs: 97-98 or 168-169.

One embodiment of the invention is an isolated polynucleotide encodingthe FN3 domain specifically binding c-Met having the amino acid sequenceof the sequence shown in SEQ ID NOs: 32-49 or 111-114.

One embodiment of the invention is an isolated polynucleotide encodingthe bispecific EGFR/-c-Met FN3 domain containing molecule having theamino acid sequence of SEQ ID NOs: 50-72, 106, 118-121 or 138-165.

One embodiment of the invention is an isolated polynucleotide comprisingthe polynucleotide sequence of SEQ ID NOs: 115-116 or 166-167.

The polynucleotides of the invention may be produced by chemicalsynthesis such as solid phase polynucleotide synthesis on an automatedpolynucleotide synthesizer and assembled into complete single or doublestranded molecules. Alternatively, the polynucleotides of the inventionmay be produced by other techniques such a PCR followed by routinecloning. Techniques for producing or obtaining polynucleotides of agiven known sequence are well known in the art.

The polynucleotides of the invention may comprise at least onenon-coding sequence, such as a promoter or enhancer sequence, intron,polyadenylation signal, a cis sequence facilitating RepA binding, andthe like. The polynucleotide sequences may also comprise additionalsequences encoding additional amino acids that encode for example amarker or a tag sequence such as a histidine tag or an HA tag tofacilitate purification or detection of the protein, a signal sequence,a fusion protein partner such as RepA, Fc or bacteriophage coat proteinsuch as pIX or pIII.

Another embodiment of the invention is a vector comprising at least onepolynucleotide of the invention. Such vectors may be plasmid vectors,viral vectors, vectors for baculovirus expression, transposon basedvectors or any other vector suitable for introduction of thepolynucleotides of the invention into a given organism or geneticbackground by any means. Such vectors may be expression vectorscomprising nucleic acid sequence elements that can control, regulate,cause or permit expression of a polypeptide encoded by such a vector.Such elements may comprise transcriptional enhancer binding sites, RNApolymerase initiation sites, ribosome binding sites, and other sitesthat facilitate the expression of encoded polypeptides in a givenexpression system. Such expression systems may be cell-based, orcell-free systems well known in the art.

Another embodiment of the invention is a host cell comprising the vectorof the invention. A monospecific EGFR-binding or c-Met binding FN3domain or bispecific EGFR/c-Met FN3 domain containing molecule of theinvention can be optionally produced by a cell line, a mixed cell line,an immortalized cell or clonal population of immortalized cells, as wellknown in the art. See, e.g., Ausubel, et al., ed., Current Protocols inMolecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001);Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2^(nd)Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane, Antibodies, aLaboratory Manual, Cold Spring Harbor, N.Y. (1989); Colligan, et al.,eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY(1994-2001); Colligan et al., Current Protocols in Protein Science, JohnWiley & Sons, NY, N.Y., (1997-2001).

The host cell chosen for expression may be of mammalian origin or may beselected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0,HeLa, myeloma, lymphoma, yeast, insect or plant cells, or anyderivative, immortalized or transformed cell thereof. Alternatively, thehost cell may be selected from a species or organism incapable ofglycosylating polypeptides, e.g. a prokaryotic cell or organism, such asBL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3),and any of the natural or engineered E. coli spp, Klebsiella spp., orPseudomonas spp strains.

Another embodiment of the invention is a method of producing theisolated FN3 domain specifically binding EGFR or c-Met of the inventionor the isolated bispecific EGFR/c-Met FN3 domain containing molecule ofthe invention, comprising culturing the isolated host cell of theinvention under conditions such that the isolated FN3 domainspecifically binding EGFR or c-Met or the isolated bispecific EGFR-c-MetFN3 domain containing molecule is expressed, and purifying the domain ormolecule.

The FN3 domain specifically binding EGFR or c-Met or the isolatedbispecific EGFR/c-Met FN3 domain containing molecule of the inventioncan be purified from recombinant cell cultures by well-known methods,for example by protein A purification, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography, or high performance liquid chromatography (HPLC).

Uses of Bispecific EGFR/c-Met FN3 Domain Containing Molecules andEGFR-Binding or c-Met Binding FN3 Domains of the Invention

The bispecific EGFR/c-Met FN3 domain containing molecules, the EGFRbinding FN3 domains or the c-Met binding FN3 domains of the inventionmay be used to diagnose, monitor, modulate, treat, alleviate, helpprevent the incidence of, or reduce the symptoms of human disease orspecific pathologies in cells, tissues, organs, fluid, or, generally, ahost. The methods of the invention may be used to treat an animalpatient belonging to any classification. Examples of such animalsinclude mammals such as humans, rodents, dogs, cats and farm animals.

One aspect of the invention is a method for inhibiting growth orproliferation of cells that express EGFR and/or c-Met, comprisingcontacting the cells with the isolated bispecific EGFR/c-Met FN3 domaincontaining molecule, the EGFR binding FN3 domain or the c-Met bindingFN3 domain of the invention.

Another aspect of the invention is a method for inhibiting growth ormetastasis of EGFR and/or c-Met-expressing tumor or cancer cells in asubject comprising administering to the subject an effective amount ofthe isolated bispecific EGFR/c-Met FN3 domain containing molecule, theEGFR binding FN3 domain or the c-Met binding FN3 domain of the inventionso that the growth or metastasis of EGFR- and/or c-Met-expressing tumoror cancer cell is inhibited.

The bispecific EGFR/c-Met FN3 domain containing molecule, the EGFRbinding FN3 domain or the c-Met binding FN3 domain of the invention maybe used for treatment of any disease or disorder characterized byabnormal activation or production of EGFR, c-Met, EGF or other EGFRligand or HGF, or disorder related to EGFR or c-Met expression, whichmay or may not involve malignancy or cancer, where abnormal activationand/or production of EGFR, c-Met, EGF or other EGFR ligand, or HGF isoccurring in cells or tissues of a subject having, or predisposed to,the disease or disorder.

The bispecific EGFR/c-Met FN3 domain containing molecule of theinvention may be used for treatment of tumors, including cancers andbenign tumors. Cancers that are amenable to treatment by the bispecificmolecules of the invention include those that overexpress EGFR and/orc-Met. Exemplary cancers that are amenable to treatment by thebispecific molecules of the invention include epithelial cell cancers,breast cancer, ovarian cancer, lung cancer, non-small cell lung cancer(NSCLC), lung adenocarcinoma, colorectal cancer, anal cancer, prostatecancer, kidney cancer, bladder cancer, head and neck cancer, ovariancancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer,vaginal cancer, cervical cancer, cancer of the spleen, testicularcancer, gastric cancer, cancer of the thymus, colon cancer, thyroidcancer, liver cancer, or sporadic or hereditary papillary renalcarcinoma (PRCC).

The FN3 domains that specifically bind c-Met and block binding of HGF toc-Met of the invention may be for treatment of tumors, including cancersand benign tumors. Cancers that are amenable to treatment by the c-Metbinding FN3 domains of the invention include those that overexpressc-Met. Exemplary cancers that are amenable to treatment by the FN3domains of the invention include epithelial cell cancers, breast cancer,ovarian cancer, lung cancer, colorectal cancer, anal cancer, prostatecancer, kidney cancer, bladder cancer, head and neck cancer, ovariancancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer,vaginal cancer, cervical cancer, cancer of the spleen, testicularcancer, and cancer of the thymus.

The FN3 domains that specifically bind EGFR and blocks binding of EGF tothe EGFR of the invention may be used for treatment of tumors, includingcancers and benign tumors. Cancers that are amenable to treatment by theFN3 domains of the invention include those that overexpress EGFR orvariants. Exemplary cancers that are amenable to treatment by the FN3domains of the invention include epithelial cell cancers, breast cancer,ovarian cancer, lung cancer, colorectal cancer, anal cancer, prostatecancer, kidney cancer, bladder cancer, head and neck cancer, ovariancancer, pancreatic cancer, skin cancer, oral cancer, esophageal cancer,vaginal cancer, cervical cancer, cancer of the spleen, testicularcancer, and cancer of the thymus.

Administration/Pharmaceutical Compositions

For therapeutic use, the bispecific EGFR/c-Met FN3 domain containingmolecules, the EGFR-binding FN3 domains or the c-Met-binding FN3 domainsof the invention may be prepared as pharmaceutical compositionscontaining an effective amount of the domain or molecule as an activeingredient in a pharmaceutically acceptable carrier. The term “carrier”refers to a diluent, adjuvant, excipient, or vehicle with which theactive compound is administered. Such vehicles can be liquids, such aswater and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. For example, 0.4% saline and 0.3% glycine can be used.These solutions are sterile and generally free of particulate matter.They may be sterilized by conventional, well-known sterilizationtechniques (e.g., filtration). The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, stabilizing, thickening, lubricating and coloring agents, etc.The concentration of the molecules of the invention in suchpharmaceutical formulation can vary widely, i.e., from less than about0.5%, usually at or at least about 1% to as much as 15 or 20% by weightand will be selected primarily based on required dose, fluid volumes,viscosities, etc., according to the particular mode of administrationselected. Suitable vehicles and formulations, inclusive of other humanproteins, e.g., human serum albumin, are described, for example, in e.g.Remington: The Science and Practice of Pharmacy, 21^(st) Edition, Troy,D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the bispecificEGFR/c-Met FN3 domain containing molecules, the EGFR binding FN3 domainsor the c-Met binding FN3 domains of the invention may be any suitableroute that delivers the agent to the host, such as parenteraladministration, e.g., intradermal, intramuscular, intraperitoneal,intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal,intravaginal, rectal); using a formulation in a tablet, capsule,solution, powder, gel, particle; and contained in a syringe, animplanted device, osmotic pump, cartridge, micropump; or other meansappreciated by the skilled artisan, as well known in the art. Sitespecific administration may be achieved by for example intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracerebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intracardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravascular, intravesical, intralesional, vaginal, rectal, buccal,sublingual, intranasal, or transdermal delivery.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 ml sterile buffered water, andbetween about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg ormore preferably, about 5 mg to about 25 mg, of the FN3 domain of theinvention. Similarly, a pharmaceutical composition of the invention forintravenous infusion could be made up to contain about 250 ml of sterileRinger's solution, and about 1 mg to about 30 mg, e.g. about 5 mg toabout 25 mg of the bispecific EGFR/c-Met FN3 domain containing molecule,the EGFR binding FN3 domain or the c-Met binding FN3 domain of theinvention. Actual methods for preparing parenterally administrablecompositions are well known and are described in more detail in, forexample, “Remington's Pharmaceutical Science”, 15th ed., Mack PublishingCompany, Easton, Pa.

The bispecific EGFR/c-Met FN3 domain containing molecules, theEGFR-binding FN3 domains or the c-Met-binding FN3 domains of theinvention can be lyophilized for storage and reconstituted in a suitablecarrier prior to use. This technique has been shown to be effective withconventional protein preparations and art-known lyophilization andreconstitution techniques can be employed.

The bispecific EGFR/c-Met FN3 domain containing molecules, theEGFR-binding FN3 domains or the c-Met-binding FN3 domains may beadministered to a subject in a single dose or the administration may berepeated, e.g. after one day, two days, three days, five days, six days,one week, two weeks, three weeks, one month, five weeks, six weeks,seven weeks, two months or three months. The repeated administration canbe at the same dose or at a different dose. The administration can berepeated once, twice, three times, four times, five times, six times,seven times, eight times, nine times, ten times, or more.

The bispecific EGFR/c-Met FN3 domain containing molecules, theEGFR-binding FN3 domains or the c-Met-binding FN3 domains may beadministered in combination with a second therapeutic agentsimultaneously, sequentially or separately. The second therapeutic agentmay be a chemotherapeutic agent, an anti-angiogenic agent, or acytotoxic drug. When used for treating cancer, the bispecific EGFR/c-MetFN3 domain containing molecules, the EGFR-binding FN3 domains or thec-Met-binding FN3 domains may be used in combination with conventionalcancer therapies, such as surgery, radiotherapy, chemotherapy orcombinations thereof. Exemplary agents that can be used in combinationwith the FN3 domains of the invention are antagonists of HER2, HER3,HER4, VEGF, and protein tyrosine kinase inhibitors such as Iressa®(gefitinib) and Tarceva (erlotinib).

While having described the invention in general terms, the embodimentsof the invention will be further disclosed in the following examplesthat should not be construed as limiting the scope of the claims.

Example 1. Construction of Tencon Libraries

Tencon (SEQ ID NO: 1) is an immunoglobulin-like scaffold, fibronectintype III (FN3) domain, designed from a consensus sequence of fifteen FN3domains from human tenascin-C (Jacobs et al., Protein Engineering,Design, and Selection, 25:107-117, 2012; U.S. Pat. Publ. No.2010/0216708). The crystal structure of Tencon shows six surface-exposedloops that connect seven beta-strands. These loops, or selected residueswithin each loop, can be randomized in order to construct libraries offibronectin type III (FN3) domains that can be used to select novelmolecules that bind to specific targets.

Tencon: (SEQ ID NO 1) LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT:Construction of TCL1 Library

A library designed to randomize only the FG loop of Tencon (SEQ ID NO:1), TCL1, was constructed for use with the cis-display system (Jacobs etal., Protein Engineering, Design, and Selection, 25:107-117, 2012). Inthis system, a single-strand DNA incorporating sequences for a Tacpromoter, Tencon library coding sequence, RepA coding sequence,cis-element, and ori element is produced. Upon expression in an in vitrotranscription/translation system, a complex is produced of theTencon-RepA fusion protein bound in cis to the DNA from which it isencoded. Complexes that bind to a target molecule are then isolated andamplified by polymerase chain reaction (PCR), as described below.

Construction of the TCL1 library for use with cis-display was achievedby successive rounds of PCR to produce the final linear, double-strandedDNA molecules in two halves; the 5′ fragment contains the promoter andTencon sequences, while the 3′ fragment contains the repA gene and thecis- and ori elements. These two halves are combined by restrictiondigest in order to produce the entire construct. The TCL1 library wasdesigned to incorporate random amino acids only in the FG loop ofTencon, KGGHRSN (SEQ ID NO: 86). NNS codons were used in theconstruction of this library, resulting in the possible incorporation ofall 20 amino acids and one STOP codon into the FG loop. The TCL1 librarycontains six separate sub-libraries, each having a different randomizedFG loop length, from 7 to 12 residues, in order to further increasediversity. Design of tencon-based libraries are shown in Table 2.

TABLE 2 Library BC Loop Design FG Loop Design WT Tencon TAPDAAFD*KGGHRSN** TCL1 TAPDAAFD* XXXXXXX XXXXXXXX XXXXXXXXX XXXXXXXXXXXXXXXXXXXXX XXXXXXXXXXXX TCL2 ######## #####S## *TAPDAAFD: residues22-28 of SEQ ID NO: 1; **KGGHRSN: SEQ ID NO: 86 Xrefers to degenerateamino acids encoded by NNS codons. #refers to the “designed distributionof amino acids” described in the text.

To construct the TCL1 library, successive rounds of PCR were performedto append the Tac promoter, build degeneracy into the F:G loop, and addnecessary restriction sites for final assembly. First, a DNA sequencecontaining the promoter sequence and Tencon sequence 5′ of the FG loopwas generated by PCR in two steps. DNA corresponding to the full Tencongene sequence was used as a PCR template with primers POP2220 (SEQID NO:2) and TC5′ to FG (SEQID NO: 3). The resulting PCR product from thisreaction was used as a template for the next round of PCR amplificationwith primers 130mer (SEQID NO: 4) and Tc5′ to FG to complete theappending of the 5′ and promoter sequences to Tencon. Next, diversitywas introduced into the F:G loop by amplifying the DNA product producedin the first step with forward primer POP2222 (SEQID NO: 5), and reverseprimers TCF7 (SEQID NO: 6), TCF8 (SEQID NO: 7), TCF9 (SEQID NO: 8),TCF10 (SEQID NO: 9), TCF11 (SEQID N NO: 10), or TCF12 (SEQID NO: 11),which contain degenerate nucleotides. At least eight 100 μL PCRreactions were performed for each sub-library to minimize PCR cycles andmaximize the diversity of the library. At least 5×g of this PCR productwere gel-purified and used in a subsequent PCR step, with primersPOP2222 (SEQ ID NO: 5) and POP2234 (SEQID NO: 12), resulting in theattachment of a 6×His tag and NotI restriction site to the 3′ end of theTencon sequence. This PCR reaction was carried out using only fifteenPCR cycles and at least 500 ng of template DNA. The resulting PCRproduct was gel-purified, digested with NotI restriction enzyme, andpurified by Qiagen column.

The 3′ fragment of the library is a constant DNA sequence containingelements for display, including a PspOMI restriction site, the codingregion of the repA gene, and the cis- and ori elements. PCR reactionswere performed using a plasmid (pCR4Blunt) (Invitrogen) containing thisDNA fragment with M13 Forward and M13 Reverse primers. The resulting PCRproducts were digested by PspOMI overnight and gel-purified. To ligatethe 5′ portion of library DNA to the 3′ DNA containing the repA gene, 2pmol of 5′ DNA were ligated to an equal molar amount of 3′ repA DNA inthe presence of NotI and PspOMI enzymes and T4 ligase. After overnightligation at 37° C., a small portion of the ligated DNA was run on a gelto check ligation efficiency. The ligated library product was split intotwelve PCR amplifications and a 12-cycle PCR reaction was run withprimer pair POP2250 (SEQID NO: 13) and DidLigRev (SEQID NO: 14). The DNAyield for each sub-library of TCL1 library ranged from 32-34 μg.

To assess the quality of the library, a small portion of the workinglibrary was amplified with primers Tcon5new2 (SEQID NO: 15) and Tcon6(SEQID NO: 16), and was cloned into a modified pET vector vialigase-independent cloning. The plasmid DNA was transformed intoBL21-GOLD (DE3) competent cells (Stratagene) and 96 randomly pickedcolonies were sequenced using a T7 promoter primer. No duplicatesequences were found. Overall, approximately 70-85% of clones had acomplete promoter and Tencon coding sequence without frame-shiftmutation. The functional sequence rate, which excludes clones with STOPcodons, was between 59% and 80%.

Construction of TCL2 Library

TCL2 library was constructed in which both the BC and FG loops of Tenconwere randomized and the distribution of amino acids at each position wasstrictly controlled. Table 3 shows the amino acid distribution atdesired loop positions in the TCL2 library. The designed amino aciddistribution had two aims. First, the library was biased toward residuesthat were predicted to be structurally important for Tencon folding andstability based on analysis of the Tencon crystal structure and/or fromhomology modeling. For example, position 29 was fixed to be only asubset of hydrophobic amino acids, as this residue was buried in thehydrophobic core of the Tencon fold. A second layer of design includedbiasing the amino acid distribution toward that of residuespreferentially found in the heavy chain HCDR3 of antibodies, toefficiently produce high-affinity binders (Birtalan et al., J Mol Biol377:1518-28, 2008; Olson et al., Protein Sci 16:476-84, 2007). Towardsthis goal, the “designed distribution” of Table 3 refers to thedistribution as follows: 6% alanine, 6% arginine, 3.9% asparagine, 7.5%aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2.3%histidine, 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenylalanine,4% proline, 10% serine, 4.5% threonine, 4% tryptophan, 17.3% tyrosine,and 4% valine. This distribution is devoid of methionine, cysteine, andSTOP codons.

TABLE 3 Residue WT Position* residues Distribution in the TCL2 library22 T designed distribution 23 A designed distribution 24 P 50% P +designed distribution 25 D designed distribution 26 A 20% A + 20% G +designed distribution 27 A designed distribution 28 F 20% F, 20% I, 20%L, 20% V, 20% Y 29 D 33% D, 33% E, 33% T 75 K designed distribution 76 Gdesigned distribution 77 G designed distribution 78 H designeddistribution 79 R designed distribution 80 S 100% S 81 N designeddistribution 82 P 50% P + designed distribution *residue numbering isbased on Tencon sequence of SEQ ID NO: 1

The 5′ fragment of the TCL2 library contained the promoter and thecoding region of Tencon (SEQ ID NO: 1), which was chemically synthesizedas a library pool (Sloning Biotechnology). This pool of DNA contained atleast 1×10¹¹ different members. At the end of the fragment, a BsaIrestriction site was included in the design for ligation to RepA.

The 3′ fragment of the library was a constant DNA sequence containingelements for display including a 6×His tag, the coding region of therepA gene, and the cis-element. The DNA was prepared by PCR reactionusing an existing DNA template (above), and primers LS1008 (SEQID NO:17) and DidLigRev (SEQID NO: 14). To assemble the complete TCL2 library,a total of 1 μg of BsaI-digested 5′ Tencon library DNA was ligated to3.5 μg of the 3′ fragment that was prepared by restriction digestionwith the same enzyme. After overnight ligation, the DNA was purified byQiagen column and the DNA was quantified by measuring absorbance at 260nm. The ligated library product was amplified by a 12-cycle PCR reactionwith primer pair POP2250 (SEQID NO: 13) and DidLigRev (SEQID NO: 14). Atotal of 72 reactions were performed, each containing 50 ng of ligatedDNA products as a template. The total yield of TCL2 working library DNAwas about 100 μg. A small portion of the working library was sub-clonedand sequenced, as described above for library TCL1. No duplicatesequences were found. About 80% of the sequences contained completepromoter and Tencon coding sequences with no frame-shift mutations.

Construction of TCL14 Library

The top (BC, DE, and FG) and the bottom (AB, CD, and EF) loops, e.g.,the reported binding surfaces in the FN3 domains are separated by thebeta-strands that form the center of the FN3 structure. Alternativesurfaces residing on the two “sides” of the FN3 domains having differentshapes than the surfaces formed by loops only are formed at one side ofthe FN3 domain by two anti-parallel beta-strands, the C and the Fbeta-strands, and the CD and FG loops, and is herein called theC-CD-F-FG surface.

A library randomizing an alternative surface of Tencon was generated byrandomizing select surface exposed residues of the C and F strands, aswell as portions of the CD and FG loops as shown in FIG. 4. A Tenconvariant, Tencon27 (SEQ ID NO: 99) having following substitutions whencompared to Tencon (SEQ ID NO: 1) was used to generate the library; E11RL17A, N46V, E86I. A full description of the methods used to constructthis library is described in U.S. patent application Ser. No.13/852,930.

Example 2: Selection of Fibronectin Type III (FN3) Domains that BindEGFR and Inhibit EGF Binding

Library Screening

Cis-display was used to select EGFR binding domains from the TCL1 andTCL2 libraries. A recombinant human extracellular domain of EGFR fusedto an IgG1 Fc (R&D Systems) was biotinylated using standard methods andused for panning (residues 25-645 of full length EGFR of SEQ ID NO: 73).For in vitro transcription and translation (ITT), 2-6 μg of library DNAwere incubated with 0.1 mM complete amino acids, 1×S30 premixcomponents, and 30 μL of S30 extract (Promega) in a total volume of 100μL and incubated at 30° C. After 1 hour, 450 μL of blocking solution(PBS pH 7.4, supplemented with 2% bovine serum albumin, 100 μg/mLherring sperm DNA, and 1 mg/mL heparin) were added and the reaction wasincubated on ice for 15 minutes. EGFR-Fc:EGF complexes were assembled atmolar ratios of 1:1 and 10:1 EGFR to EGF by mixing recombinant human EGF(R&D Systems) with biotinylated recombinant EGFR-Fc in blocking solutionfor 1 hour at room temperature. For binding, 500 μL of blocked ITTreactions were mixed with 100 μL of EGFR-Fc:EGF complexes and incubatedfor 1 hour at room temperature, after which bound complexes were pulleddown with magnetic neutravidin or streptavidin beads (Seradyne). Unboundlibrary members were removed by successive washes with PBST and PBS.After washing, DNA was eluted from the bound complexes by heating to 65°C. for 10 minutes, amplified by PCR, and attached to a DNA fragmentencoding RepA by restriction digestion and ligation for further roundsof panning. High affinity binders were isolated by successively loweringthe concentration of target EGFR-Fc during each round from 200 nM to 50nM and increasing the washing stringency. In rounds 4 and 5, unbound andweakly bound FN3 domains were removed by washing in the presence of a10-fold molar excess of non-biotinylated EGFR-Fc overnight in PBS.

Following panning, selected FN3 domains were amplified by PCR usingoligos Tcon5new2 (SEQID NO: 15) and Tcon6 (SEQID NO: 16), subcloned intoa pET vector modified to include a ligase independent cloning site, andtransformed into BL21-GOLD (DE3) (Stratagene) cells for solubleexpression in E. coli using standard molecular biology techniques. Agene sequence encoding a C-terminal poly-histidine tag was added to eachFN3 domain to enable purification and detection. Cultures were grown toan optical density of 0.6-0.8 in 2YT medium supplemented with 100 μg/mLcarbenicillin in 1-mL 96-well blocks at 37° C. before the addition ofIPTG to 1 mM, at which point the temperature was reduced to 30° C. Cellswere harvested approximately 16 hours later by centrifugation and frozenat −20° C. Cell lysis was achieved by incubating each pellet in 0.6 mLof BugBuster® HT lysis buffer (Novagen EMD Biosciences) with shaking atroom temperature for 45 minutes.

Selection of FN3 Domains that Bind EGFR on Cells

To assess the ability of different FN3 domains to bind EGFR in a morephysiological context, their ability to bind A431 cells was measured.A431 cells (American Type Culture Collection, cat. # CRL-1555)over-express EGFR with ˜2×10⁶ receptors per cell. Cells were plated at5,000/well in opaque black 96-well plates and allowed to attachovernight at 37° C., in a humidified 5% CO₂ atmosphere. FN3domain-expressing bacterial lysates were diluted 1,000-fold into FACSstain buffer (Becton Dickinson) and incubated for 1 hour at roomtemperature in triplicate plates. Lysates were removed and cells werewashed 3 times with 150 μL/well of FACS stain buffer. Cells wereincubated with 50 μL/well of anti-penta His-Alexa488 antibody conjugate(Qiagen) diluted 1:100 in FACS stain buffer for 20 minutes at roomtemperature. Cells were washed 3 times with 150 μL/well of FACS stainbuffer, after which wells were filled with 100 μL of FACS stain bufferand read for fluorescence at 488 nm using an Acumen eX3 reader.Bacterial lysates containing FN3 domains were screened for their abilityto bind A431 cells (1320 crude bacterial lysates for TCL1 and TCL2libraries) and 516 positive clones were identified, where binding was≥10-fold over the background signal. 300 lysates from the TCL14 librarywere screened for binding, resulting in 58 unique FN3 domain sequencesthat bind to EGFR.

Selection of FN3 Domains that Inhibit EGF Binding to EGFR on Cells

To better characterize the mechanism of EGFR binding, the ability ofvarious identified FN3 domain clones to bind EGFR in an EGF-competitivemanner was measured using A431 cells and run in parallel with the A431binding assay screen. A431 cells were plated at 5,000/well in opaqueblack 96-well plates and allowed to attach overnight at 37° C., in ahumidified 5% CO₂ atmosphere. Cells were incubated with 50 μL/well of1:1,000 diluted bacterial lysate for 1 hour at room temperature intriplicate plates. Biotinylated EGF (Invitrogen, cat. # E-3477) wasadded to each well to give a final concentration of 30 ng/mL andincubated for 10 minutes at room temperature. Cells were washed 3 timeswith 150 μL/well of FACS stain buffer. Cells were incubated with 50μL/well of streptavidin-phycoerythrin conjugate (Invitrogen) diluted1:100 in FACS stain buffer for 20 minutes at room temperature. Cellswere washed 3 times with 150 μL/well of FACS stain buffer, after whichwells were filled with 100 μL of FACS stain buffer and read forfluorescence at 600 nm using an Acumen eX3 reader.

Bacterial lysates containing the FN3 domains were screened in the EGFcompetition assay described above. 1320 crude bacterial lysates fromTCL1 and TCL2 libraries were screened resulting in 451 positive clonesthat inhibited EGF binding by >50%.

Expression and Purification of Identified FN3 Domains Binding EGFR

His-tagged FN3 domains were purified from clarified E. coli lysates withHis MultiTrap™ HP plates (GE Healthcare) and eluted in buffer containing20 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole atpH 7.4. Purified samples were exchanged into PBS pH 7.4 for analysisusing PD MultiTrap™ G-25 plates (GE Healthcare).

Size Exclusion Chromatography Analysis

Size exclusion chromatography was used to determine the aggregationstate of the FN3 domains binding EGFR. Aliquots (10 μL) of each purifiedFN3 domain were injected onto a Superdex 75 5/150 column (GE Healthcare)at a flow rate of 0.3 mL/min in a mobile phase of PBS pH 7.4. Elutionfrom the column was monitored by absorbance at 280 nm. Centyrins thatexhibited high levels of aggregation by SEC were excluded from furtheranalysis.

Off-Rate of Selected EGFR-Binding FN3 Domains from EGFR-Fc

Select EGFR-binding FN3 domains were screened to identify those withslow off-rates (k_(off)) in binding to EGFR-Fc on a ProteOn XPR-36instrument (Bio-Rad) to facilitate selection of high affinity binders.Goat anti-human Fc IgG (R&D systems), at a concentration of 5 μg/mL, wasdirectly immobilized via amine coupling (at pH 5.0) on all 6 ligandchannels in horizontal orientation on the chip with a flow rate of 30μL/min in PBS containing 0.005% Tween-20. The immobilization densitiesaveraged about 1500 Response Units (RU) with less than 5% variationamong different channels. EGFR-Fc was captured on the anti-human Fc IgGsurface to a density around 600 RU in vertical ligand orientation. Alltested FN3 domains were normalized to a concentration of 1 μM and testedfor their binding in horizontal orientation. All 6 analyte channels wereused for the FN3 domains to maximize screening throughput. Thedissociation phase was monitored for 10 minutes at a flow rate of 100μL/min. The inter-spot binding signals were used as references tomonitor non-specific binding between analytes and the immobilized IgGsurface, and were subtracted from all binding responses. The processedbinding data were locally fit to a 1:1 simple Langmuir binding model toextract the k_(off) for each FN3 domain binding to captured EGFR-Fc.

Inhibition of EGF-Stimulated EGFR Phosphorylation

Purified EGFR-binding FN3 domains were tested for their ability toinhibit EGF-stimulated phosphorylation of EGFR in A431 cells at a singleconcentration. EGFR phosphorylation was monitored using the EGFRphospho(Tyr1173) kit (Meso Scale Discovery). Cells were plated at20,000/well in clear 96-well tissue culture-treated plates (Nunc) in 100μL/well of RPMI medium (Gibco) containing GlutaMAX™ with 10% fetalbovine serum (FBS) (Gibco) and allowed to attach overnight at 37° C. ina humidified 5% CO₂ atmosphere. Culture medium was removed completelyand cells were starved overnight in 100 μL/well of medium containing noFBS at 37° C. in a humidified 5% CO₂ atmosphere. Cells were then treatedwith 100 μL/well of pre-warmed (37° C.) starvation medium containingEGFR-binding FN3 domains at a concentration of 2 μM for 1 hour at 37° C.in a humidified 5% CO₂ atmosphere. Controls were treated with starvationmedium only. Cells were stimulated by the addition and gentle mixing of100 μL/well of pre-warmed (37° C.) starvation medium containing 100ng/mL recombinant human EGF (R&D Systems, cat. #236-EG), for finalconcentrations of 50 ng/mL EGF and 1 μM EGFR-binding FN3 domain, andincubation at 37° C., 5% CO₂ for 15 minutes. One set of control wellswas left un-stimulated as negative controls. Medium was completelyremoved and cells were lysed with 100 μL/well of Complete Lysis Buffer(Meso Scale Discovery) for 10 minutes at room temperature with shaking,as per the manufacturer's instructions. Assay plates configured formeasuring EGFR phosphorylated on tyrosine 1173 (Meso Scale Discovery)were blocked with the provided blocking solution as per themanufacturer's instructions at room temperature for 1.5-2 hours. Plateswere then washed 4 times with 200 μL/well of 1× Tris Wash Buffer (MesoScale Discovery). Aliquots of cell lysate (30 μL/well) were transferredto assay plates, which were covered with plate sealing film (VWR) andincubated at room temperature with shaking for 1 hour. Assay plates werewashed 4 times with 200 μL/well of Tris Wash Buffer, after which 25 μLof ice-cold Detection Antibody Solution (Meso Scale Discovery) wereadded to each well, being careful not to introduce bubbles. Plates wereincubated at room temperature with shaking for 1 hour, followed by 4washes with 200 μL/well of Tris Wash Buffer. Signals were detected byaddition of 150 μL/well of Read Buffer (Meso Scale Discovery) andreading on a SECTOR® Imager 6000 instrument (Meso Scale Discovery) usingmanufacturer-installed assay-specific default settings. Percentinhibition of the EGF-stimulated positive control signal was calculatedfor each EGFR-binding FN3 domain.

Inhibition of EGF-stimulated EGFR phosphorylation was measured for 232identified clones from the TCL1 and TCL2 libraries. 22 of these clonesinhibited EGFR phosphorylation by ≥50% at 1 μM concentration. Afterremoval of clones that either expressed poorly or were judged to bemultimeric by size exclusion chromatography, nine clones were carriedforward for further biological characterization. The BC and FG loopsequences of these clones are shown in Table 4. Eight of the nineselected clones had a common FG loop sequence (HNVYKDTNMRGL; SEQ ID NO:95) and areas of significant similarity were seen between several clonesin their BC loop sequences.

TABLE 4 FN3 Domain BC Loop FG Loop SEQ ID SEQ ID SEQ ID Clone ID NO:Sequence NO: Sequence NO: P53A1R5- 18 ADPHGFYD 87 HNVYKDTNMRGL 95 17P54AR4-17 19 TYDRDGYD 88 HNVYKDTNMRGL 95 P54AR4-47 20 WDPFSFYD 89HNVYKDTNMRGL 95 P54AR4-48 21 DDPRGFYE 90 HNVYKDTNMRGL 95 P54AR4-73 22TWPYADLD 91 HNVYKDTNMRGL 95 P54AR4-74 23 GYNGDHFD 92 HNVYKDTNMRGL 95P54AR4-81 24 DYDLGVYD 93 HNVYKDTNMRGL 95 P54AR4-83 25 DDPWDFYE 94HNVYKDTNMRGL 95 P54CR4-31 26 TAPDAAFD 85 LGSYVFEHDVM 96

Example 3: Characterization of EGFR-Binding FN3 Domains that Inhibit EGFBinding

Large-scale Expression, Purification, and Endotoxin Removal

The 9 FN3 domains shown in Table 4 were scaled up to provide morematerial for detailed characterization. An overnight culture containingeach EGFR-binding FN3 domain variant was used to inoculate 0.8 L ofTerrific broth medium supplemented with 100 μg/mL ampicillin at a 1/80dilution of overnight culture into fresh medium, and incubated withshaking at 37° C. The culture was induced when the optical density at600 nm reached ˜1.2-1.5 by addition of IPTG to a final concentration of1 mM and the temperature was reduced to 30° C. After 4 hours, cells werecollected by centrifugation and the cell pellet was stored at −80° C.until needed.

For cell lysis, the thawed pellet was resuspended in 1× BugBuster®supplemented with 25 U/mL Benzonase® (Sigma-Aldrich) and 1 kU/mLrLysozyme™ (Novagen EMD Biosciences) at a ratio of 5 mL of BugBuster®per gram of pellet. Lysis proceeded for 1 hour at room temperature withgentle agitation, followed by centrifugation at 56,000×g for 50 minutesat 4° C. The supernatant was collected and filtered through a 0.2 unfilter, then loaded on to a 5-mL HisTrap FF column pre-equilibrated withBuffer A (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 10 mM imidazole) using aGE Healthcare ÄKTAexplorer 100s chromatography system. The column waswashed with 20 column volumes of Buffer A and further washed with 16%Buffer B (50 mM Tris-HCl pH7.5, 500 mM NaCl, 250 mM imidazole) for 6column volumes. The FN3 domains were eluted with 50% B for 10 columnvolumes, followed by a gradient from 50-100% B over 6 column volumes.Fractions containing the FN3 domain protein were pooled, concentratedusing a Millipore 10K MWCO concentrator, and filtered before loadingonto a HiLoad™ 16/60 Superdex™ 75 column (GE Healthcare)pre-equilibrated with PBS. The protein monomer peak eluting from thesize exclusion column was retained.

Endotoxins were removed using a batch approach with ActiClean Etox resin(Sterogene Bioseparations). Prior to endotoxin removal, the resin waspre-treated with 1 N NaOH for 2 hours at 37° C. (or overnight at 4° C.)and washed extensively with PBS until the pH had stabilized to ˜7 asmeasured with pH indicator paper. The purified protein was filteredthrough a 0.2 μm filter before adding to 1 mL of Etox resin at a ratioof 10 mL of protein to 1 mL of resin. The binding of endotoxin to resinwas allowed to proceed at room temperature for at least 2 hours withgentle rotation. The resin was removed by centrifugation at 500×g for 2minutes and the protein supernatant was retained. Endotoxin levels weremeasured using EndoSafe®-PTS™ cartridges and analyzed on anEndoSafe®-MCS reader (Charles River). If endotoxin levels were above 5EU/mg after the first Etox treatment, the above procedure was repeateduntil endotoxin levels were decreased to ≤5 EU/mg. In cases where theendotoxin level was above 5 EU/mg and stabilized after two consecutivetreatments with Etox, anion exchange or hydrophobic interactionchromatography conditions were established for the protein to remove theremaining endotoxins.

Affinity Determination of Selected EGFR-Binding FN3 Domains to EGFR-Fc(EGFR-Fc Affinity)

Binding affinity of selected EGFR-binding FN3 domains to recombinantEGFR extracellular domain was further characterized by surface Plasmonresonance methods using a Proteon Instrument (BioRad). The assay set-up(chip preparation, EGFR-Fc capture) was similar to that described abovefor off-rate analysis. Selected EGFR binding FN3 domains were tested at1 μM concentration in 3-fold dilution series in the horizontalorientation. A buffer sample was also injected to monitor the baselinestability. The dissociation phase for all concentrations of eachEGFR-binding FN3 domain was monitored at a flow rate of 100 μL/min for30 minutes (for those with k_(off)˜10⁻² s⁻¹ from off-rate screening), or1 hour (for those with k_(off)˜10⁻³ s⁻¹ or slower). Two sets ofreference data were subtracted from the response data: 1) the inter-spotsignals to correct for the non-specific interactions between theEGFR-binding FN3 domain and the immobilized IgG surface; 2) the bufferchannel signals to correct for baseline drifting due to the dissociationof captured EGFR-Fc surface over time. The processed binding data at allconcentrations for each FN3 domain were globally fit to a 1:1 simpleLangmuir binding model to extract estimates of the kinetic (k_(on),k_(off)) and affinity (K_(D)) constants. Table 5 shows the kineticconstants for each of the constructs, with the affinity varying from 200pM to 9.6 nM.

Binding of Selected EGFR-Binding FN3 Domains to EGFR on Cells (A431 CellBinding Assay)

A431 cells were plated at 5,000/well in opaque black 96-well plates andallowed to attach overnight at 37° C., in a humidified 5% CO₂atmosphere. Purified EGFR-binding FN3 domains (1.5 nM to 30 μM) wereadded to the cells (in 50 uL) for 1 hour at room temperature intriplicate plates. Supernatant was removed and cells were washed 3 timeswith 150 μL/well of FACS stain buffer. Cells were incubated with 50μL/well of anti-penta His-Alexa488 antibody conjugate (Qiagen) diluted1:100 in FACS stain buffer for 20 minutes at room temperature. Cellswere washed 3 times with 150 μL/well of FACS stain buffer, after whichwells were filled with 100 μL of FACS stain buffer and read forfluorescence at 488 nm using an Acumen eX3 reader. Data were plotted asraw fluorescence signal against the logarithm of the FN3 domain molarconcentration and fitted to a sigmoidal dose-response curve withvariable slope using GraphPad Prism 4 (GraphPad Software) to calculateEC₅₀ values. Table 5 reports the EC₅₀ for each of the constructs rangingfrom 2.2 to >20 μM.

Inhibition of EGF Binding to EGFR on Cells Using Selected EGFR-BindingFN3 Domains (A431 Cell EGF Competition Assay)

A431 cells were plated at 5,000/well in opaque black 96-well plates andallowed to attach overnight at 37° C., in a humidified 5% CO₂atmosphere. Purified EGFR-binding FN3 domains (1.5 nM to 30 μM) wereadded to the cells (50 μL/well) for 1 hour at room temperature intriplicate plates. Biotinylated EGF (Invitrogen, Cat #: E-3477) wasadded to each well to give a final concentration of 30 ng/mL andincubated for 10 minutes at room temperature. Cells were washed 3 timeswith 150 μL/well of FACS stain buffer. Cells were incubated with 50μL/well of streptavidin-phycoerythrin conjugate (Invitrogen) diluted1:100 in FACS stain buffer for 20 minutes at room temperature. Cellswere washed 3 times with 150 μL/well of FACS stain buffer, after whichwells were filled with 100 μL of FACS stain buffer and read forfluorescence at 600 nm using an Acumen eX3 reader. Data were plotted asthe raw fluorescence signal against the logarithm of FN3 domain molarconcentration and fitted to a sigmoidal dose-response curve withvariable slope using GraphPad Prism 4 (GraphPad Software) to calculateIC₅₀ values. Table 5 reports the IC₅₀ values ranging from 1.8 to 121 nM.

Inhibition of EGF-Stimulated EGFR Phosphorylation (Phoshpo-EGRF Assay)

Select FN3 domains that significantly inhibited EGF-stimulated EGFRphosphorylation were assessed more completely by measuring IC₅₀ valuesfor inhibition. Inhibition of EGF-stimulated EGFR phosphorylation wasassessed at varying FN3 domain concentrations (0.5 nM to 10 μM) asdescribed above in “inhibition of EGF stimulated EGFR phosphorylation”.Data were plotted as electrochemiluminescence signal against thelogarithm of the FN3 domain molar concentration and IC₅₀ values weredetermined by fitting data to a sigmoidal dose response with variableslope using GraphPad Prism 4 (GraphPad Software). Table 5 reports theIC₅₀ values ranging from 18 nM to >2.5 μM.

Inhibition of Human Tumor Cell Growth (NCI-H292 Growth and NCI-H322Growth Assay)

Inhibition of EGFR-dependent cell growth was assessed by measuringviability of the EGFR over-expressing human tumor cell lines, NCI-H292and NCI-H322 (American Type Culture Collection, cat. # CRL-1848 & #CRL-5806, respectively), following exposure to EGFR-binding FN3 domains.Cells were plated at 500 cells/well (NCI-H292) or 1,000 cells/well(NCI-H322) in opaque white 96-well tissue culture-treated plates (Nunc)in 100 μL/well of RPMI medium (Gibco) containing GlutaMAX™ and 10 mMHEPES, supplemented with 10% heat inactivated fetal bovine serum (Gibco)and 1% penicillin/streptomycin (Gibco), and allowed to attach overnightat 37° C. in a humidified 5% CO₂ atmosphere. Cells were treated byaddition of 5 μL/well of phosphate-buffered saline (PBS) containing aconcentration range of EGFR-binding FN3 domains. Controls were treatedwith 5 μL/well of PBS only or 25 mM ethylenediaminetetraacetic acid inPBS. Cells were incubated at 37° C., 5% CO₂ for 120 hours. Viable cellswere detected by addition of 75 μL/well of CellTiter-Glo® reagent(Promega), followed by mixing on a plate shaker for 2 minutes, andincubation in the dark at room temperature for a further 10 minutes.Plates were read on a SpectraMax M5 plate reader (Molecular Devices) setto luminescence mode, with a read time of 0.5 seconds/well against ablank of medium only. Data were plotted as a percentage of PBS-treatedcell growth against the logarithm of FN3 domain molar concentration.IC₅₀ values were determined by fitting data to the equation for asigmoidal dose response with variable slope using GraphPad Prism 4(GraphPad Software). Table 5 shows IC₅₀ values ranging from 5.9 nM to1.15 μM and 9.2 nM to >3.1 μM, using the NCI-H292 and NCI-H322 cellsrespectively.

TABLE 5 A431 EGFR- Cell A431 Phospho- NCI- NCI- FN3 SEQ Fc Binding CellEGF EGFR H292 H322 Domain ID Affinity EC₅₀ Competition IC₅₀ GrowthGrowth Clone ID NO: (nM) (nM) IC₅₀ (nM) (nM) IC₅₀ (nM) IC₅₀ (nM)P53A1R5- 18 1.89 4.0 9.8 >2500 86 65 17 P54AR4-17 19 9.62 16 21 184 NDND P54AR4-47 20 2.51 8.6 7.1 295 44 39 P54AR4-48 21 7.78 12 9.8 170 NDND P54AR4-73 22 0.197 9.4 4.6 141 83 73 P54AR4-74 23 ND 77 ND ND ND NDP54AR4-81 24 ND 84 121 ND ND ND P54AR4-83 25 0.255 2.2 1.8 18 5.9 9.2P54CR4-31 26 0.383 >20000 55 179 1150 >3073

Example 4: Engineering of EGFR-Binding FN3 Domains

A subset of the EGFR binding FN3 domains was engineered to increase theconformational stability of each molecule. The mutations L17A, N46V, andE86I (described in US Pat. Publ. No. 2011/0274623) were incorporatedinto clones P54AR4-83, P54CR4-31, and P54AR4-37 by DNA synthesis. Thenew mutants, P54AR4-83v2, P54CR431-v2, and P54AR4-37v2 were expressedand purified as described above. Differential scanning calorimetry inPBS was used to assess the stability of each mutant in order to compareit to that of the corresponding parent molecule. Table 6 shows that eachclone was stabilized significantly, with an average increase in theT_(m) of 18.5° C.

TABLE 6 FN3 domain Clone SEQ ID NO: T_(m) (° C.) P54AR4-83 25 50.6P54AR4-83v2 27 69.8 P54CR4-31 26 60.9 P54CR4-31v2 28 78.9 P54AR4-37 2245.9 P54AR4-37v2 29 64.2

Example 5: Cysteine Engineering and Chemical Conjugation of EGFR-BindingFN3 Domains

Cysteine mutants of FN3 domains are made from the base Tencon moleculeand variants thereof that do not have cysteine residues. These mutationsmay be made using standard molecular biology techniques known in the artto incorporate a unique cysteine residue into the base Tencon sequence(SEQ ID NO: 1) or other FN3 domains in order to serve as a site forchemical conjugation of small molecule drugs, fluorescent tags,polyethylene glycol, or any number of other chemical entities. The siteof mutation to be selected should meet certain criteria. For example,the Tencon molecule mutated to contain the free cysteine should: (i) behighly expressed in E. coli, (ii) maintain a high level of solubilityand thermal stability, and (iii) maintain binding to the target antigenupon conjugation. Since the Tencon scaffold is only ˜90-95 amino acids,single-cysteine variants can easily be constructed at every position ofthe scaffold to rigorously determine the ideal position(s) for chemicalconjugation.

Each individual amino acid residue, from positions 1-95 (or 2-96 whenthe N-terminal methionine is present) of the P54AR4-83v2 mutant (SEQ IDNO: 27), which binds EGFR, was mutated to cysteine to assess the bestchemical conjugation sites.

Construction, Expression and Purification

The amino acid sequence of each individual cysteine variant ofP54AR4-83v2 was reverse translated into nucleic acid sequences encodingthe proteins using preferred codons for E. coli expression and asynthetic gene was produced (DNA 2.0). These genes were cloned into apJexpress401 vector (DNA 2.0) for expression driven by a T5 promotersequence and transformed into E. coli strain BL21 (Agilent). TheP54AR4-83v2 “cys scan” library was provided as glycerol stocks arrayedinto a 96-well plate and the expression and purification of eachfollowed the same procedure described in Example 2.

Chemical Conjugation

For the P54AR4-83v2 “cys scan” library, conjugation was integrated intothe purification process. Cysteine variants in clarified lysate werebound to Ni-NTA resin in 96-well format using His-trap HP plates(catalog #28-4008-29, GE Healthcare) by adding lysate to the wells andcentrifugation at 100×g for 5 min. The resin was washed 3 times withbuffer A, and then N-ethyl maleimide (NEM) was added as 500 μL of a 1.5mM solution. Following a one-hour room temperature incubation on arotisserie shaker, excess NEM was removed by centrifugation and threewashes with buffer A. Conjugated cysteine variants was eluted with 2×150μL of buffer B and exchanged into PBS with MultiScreen Filter Plateswith Ultracel-10 membrane (catalog # MAUF1010, Millipore) or with96-well PD MultiTrap plates (catalog #28-9180-06, GE Healthcare).Conjugates were characterized by mass spectrometry (Table 7). Cysteinevariants that expressed poorly (less than 0.1 mg of protein obtainedfrom a 5 mL culture or no protein detected by mass spectrometry) orconjugated poorly to NEM (less than 80% conjugated, as determined bymass spectrometry) were excluded from further analysis. This eliminatedL1C, W21C, Q36C, E37C, A44C, D57C, L61C, Y67C, and F92C due to poorexpression and A17C, L19C, 133C, Y35C, Y56C, L58C, T65C, V69C, 171C, andT94C due to low conjugation efficiency.

TABLE 7 Cysteine Variant of Protein Yield P54AR4-83v2 (mg) ConjugationL1C 0.58 no protein detected P2C 0.28 yes A3C 1.05 yes P4C 0.77 yes K5C0.19 yes N6C 0.56 yes L7C 0.96 yes V8C 1.40 yes V9C 0.92 yes S10C 0.91yes E11C 0.82 yes V12C 0.76 yes T13C 0.53 yes E14C 1.05 yes D15C 1.12yes S16C 0.65 yes A17C 0.70 no R18C 1.14 yes L19C 0.47 no S20C 1.02 yesW21C 0.09 no protein D22C 0.80 yes D23C 0.90 yes P24C 0.63 yes W25C 1.24yes A26C 1.34 yes F27C 0.92 yes Y28C 1.15 yes E29C 1.10 yes S30C 0.80yes F31C 0.75 yes L32C 0.64 yes I33C 0.09 no Q34C 1.14 yes Y35C 0.85 noQ36C 0.04 no protein E37C 0.84 no protein S38C 0.80 yes E39C 0.72 yesK40C 1.20 yes V41C 0.99 yes G42C 1.27 yes E43C 0.22 yes A44C 0.07 yesI45C 1.14 yes V46C 0.14 yes L47C 1.12 yes T48C 1.22 yes V49C 1.10 yesP50C 0.69 yes G51C 1.15 yes S52C 0.24 yes E53C 1.13 yes R54C 1.55 yesS55C 0.88 yes Y56C 1.71 no D57C 0.09 no protein L58C 0.59 no T59C 0.80yes G60C 1.24 yes L61C 0.05 no protein K62C 1.12 yes P63C 1.44 yes G64C1.30 yes T65C 0.90 no E66C 0.20 yes Y67C 0.06 no protein T68C 0.76 yesV69C 0.62 no S70C 0.59 yes I71C 0.77 no Y72C 1.22 G73C 0.83 yes V74C0.52 yes H75C 0.55 yes N76C 1.10 yes V77C 1.12 yes Y78C 1.29 yes K79C0.29 yes D80C 1.23 yes T81C 0.59 yes N82C 0.14 yes M83C 1.03 yes R84C1.40 yes G85C 1.17 yes L86C 0.52 yes P87C 1.53 yes L88C 1.68 yes S89C1.20 yes A90C 0.71 yes I91C 0.64 yes F92C 0.05 no protein T93C 0.64 yesT94C 0.26 ~50% conjugated G95C 0.88 yes 83v2His₆-cys 1.28 yes (SEQ IDNOs: 217 and 255)Analytical Size-Exclusion Chromatography

Size exclusion chromatography for each NEM-conjugated cysteine variantsof P54AR4-83v2 was performed as described in Example 2. Table 8summarizes the results. The percent monomer for each protein wasdetermined by integrating the Abs280 signal and comparing the peak inthe monomer region (5.5-6 minutes) to the peaks in the oligomer region(4-5.3 minutes).

TABLE 8 Cysteine Variant of Percent P54AR4-83v2 monomer L1C 100 P2C 86A3C 100 P4C 100 K5C 100 N6C 94 L7C 93 V8C 91 V9C double peak S10C 80E11C 100 V12C 66 T13C 82 E14C 96 D15C 97 S16C 75 A17C 93 R18C 93 L19C 83S20C 94 W21C no protein D22C 85 D23C 100 P24C 88 W25C 76 A26C 95 F27C 97Y28C 92 E29C 85 S30C 94 F31C 57 L32C 100 I33C 100 Q34C 97 Y35C 100 Q36C100 E37C 87 S38C 93 E39C 100 K40C 97 V41C 98 G42C 87 E43C 100 A44C 100I45C 97 V46C 100 L47C 100 T48C 90 V49C 88 P50C 100 G51C 96 S52C 100 E53C97 R54C 96 S55C 100 Y56C 97 D57C 100 L58C 67 T59C 100 G60C 100 L61C noprotein K62C 95 P63C 92 G64C 100 T65C 83 E66C 100 Y67C no protein T68C100 V69C 90 S70C 100 I71C double peak Y72C 100 G73C 66 V74C 100 H75C 100N76C 94 V77C 92 Y78C 90 K79C 100 D80C 79 T81C 86 N82C 100 M83C 91 R84C100 G85C 95 L86C 83 P87C 98 L88C 98 S89C 96 A90C 100 I91C 100 F92C noprotein T93C 100 T94C 100 G95C 100 83v2His₆-cys 97 (SEQ ID NOs: 217 and255)EGFR Binding Assay

Relative binding affinity of the NEM-conjugated cysteine variants ofP54AR4-83v2 to EGFR was assessed as described in Example 2. Table 9summarizes the data showing the ratios of each cysteine variant EGFRbinding affinity relative to the P54AR4-83v2 parent protein. Cysteineconjugates that had reduced binding to EGFR (<65% of the signal observedwith P54AR4-83v2 parent when treated with 10 nM protein) as determinedby the ELISA assay were excluded from further analysis: P2C, A3C, P4C,K5C, L7C, D23C, W25C, F27C, Y28C, F31C, S55C, G73C, H75C, V77C, Y78C,T81C, N82C, M83C, and G85C.

TABLE 9 Cysteine Amount of Variant Variant of in Assay: P54AR4-83v2 500nM 100 nM 10 nM P2C 0.01 0.00 0.00 A3C 0.82 0.88 0.34 P4C 0.12 0.02 0.02K5C 0.92 1.06 0.61 N6C 0.89 1.01 0.76 L7C 0.90 1.00 0.35 V8C 0.90 1.030.96 V9C 0.93 1.03 0.94 S10C 0.96 1.07 0.83 E11C 0.95 1.08 0.90 V12C0.93 1.06 0.87 T13C 0.90 1.04 0.87 E14C 1.15 1.27 1.11 D15C 0.97 1.090.98 S16C 0.63 1.05 0.88 R18C 0.94 1.05 0.86 S20C 0.91 1.05 0.81 D22C0.90 1.02 0.84 D23C 0.40 0.20 0.02 P24C 0.83 0.85 0.45 W25C 0.70 0.640.38 A26C 0.95 1.06 0.95 F27C 0.23 0.07 0.00 Y28C 0.09 0.01 0.00 E29C0.93 1.07 0.89 S30C 0.90 1.02 0.90 F31C 0.62 0.34 0.04 L32C 0.91 1.010.87 Q34C 0.94 1.03 0.89 S38C 0.82 0.93 0.80 E39C 0.90 1.00 0.90 K40C0.86 0.95 0.88 V41C 0.95 0.99 0.92 G42C 0.90 0.99 0.84 E43C 0.92 1.040.68 I45C 0.93 1.04 0.91 V46C 0.90 1.01 0.61 L47C 0.91 1.02 0.92 T48C0.93 1.00 0.88 V49C 0.98 1.01 0.96 P50C 0.97 1.05 0.91 G51C 0.92 1.030.88 S52C 0.93 1.03 0.78 E53C 0.91 1.02 0.91 R54C 0.93 1.01 0.89 S55C0.11 0.00 0.00 T59C 0.93 1.04 0.83 G60C 0.93 1.02 0.86 K62C 0.61 0.730.64 P63C 0.92 1.02 0.95 G64C 1.36 1.42 1.28 E66C ND ND ND T68C 0.951.04 0.83 S70C 0.93 1.01 0.86 Y72C 0.93 1.00 0.93 G73C 0.21 0.02 0.00V74C 0.95 1.01 0.76 H75C 0.25 0.19 0.07 N76C 0.91 0.97 0.75 V77C 0.030.00 0.03 Y78C 0.68 0.63 0.31 K79C 0.93 0.99 0.90 D80C 0.91 0.97 0.70T81C 1.02 0.90 0.50 N82C 0.96 0.97 0.56 M83C 0.24 0.04 0.07 R84C 0.981.04 0.91 G85C 0.29 0.02 0.19 L86C 0.92 0.96 0.77 P87C 0.91 0.93 0.73L88C 0.97 1.03 0.95 S89C 1.04 1.02 0.97 A90C 1.01 1.05 0.94 I91C 1.001.01 0.90 T93C 1.04 1.05 0.96 G95C 1.00 1.03 1.01 83v2His₆-cys 1.00 1.001.00 (SEQ ID NOs: 217 and 255)Thermal Stability

The thermal stability of cysteine-NEM conjugates was assessed bydifferential scanning calorimetry (DSC). The only the conjugates testedwere those determined to express at high levels, conjugate efficiently,and retain EGFR binding. Additionally, cysteine variants within the BCand FG loops were excluded. Stability data was generated by heating a400 μL aliquot of the variant from 25° C. to 100° C. at a scan rate of1° C. per minute in a VP-DSC instrument (MicroCal). A second identicalscan was completed on the sample in order to assess the reversibility ofthermal folding/unfolding. Data was fitted to a 2-state unfolding modelin order to calculate the melting temperature (Table 10). Cys variantswith reduced melting temperatures (≤63° C., or >8° C. below theP54AR4-83v2 parent) or that demonstrated irreversible unfolding wereexcluded from further analysis: V9C, V12C, T13C, R18C, E29C, E39C, G42C,V49C, P50C, G51C, P63C.

TABLE 10 Cysteine Variant of First Scan Second Scan P54AR4-83v2 Tm (°C.) Tm (° C.) Reversible? N6C 71 70 Y V8C 69 69 Y V9C 46 46 N S10C 68 68Y E11C 71 72 Y V12C 58 58 Y T13C 63 63 Y E14C 70 71 Y D15C 73 73 Y S16C68 68 Y R18C 62 62 Y S20C 70 70 Y E29C 63 66 Y S30C 71 71 Y L32C 71 70 YQ34C 75 74 Y S38C 65 65 Y E39C 67 69 N K40C 70 70 Y V41C 71 71 Y G42C 6567 N I45C 69 68 Y L47C 67 67 Y T48C 72 72 Y V49C 54 55 N P50C 63 65 NG51C 61 61 Y E53C 76 75 Y R54C 65 65 Y T59C 67 67 Y G60C 66 66 Y K62C 6565 Y P63C 60 62 N G64C 70 70 Y T68C 72 72 Y S70C 73 72 Y Y72C 70 69 YV74C 68 67 Y L88C 70 70 Y S89C 72 71 Y A90C 67 67 Y I91C 70 69 Y T93C 6969 Y 83v2His₆-cys (SEQ 71 71 Y ID NOs: 217 and 255) P54AR4-83v2 (SEQ 7171 Y ID NO: 27)Cytotoxicity Assay

P54AR4-83v2 cysteine variants were conjugated to the cytotoxic tubulininhibitor momomethyl auristatin F (MMAF) via an enzyme-cleavable Val-Citlinker or a non-cleavable PEG₄ linker (VC-MMAF; see FIG. 2) using themethodology described for the NEM conjugation. The 32 variants thatremained after exclusions at the previous steps were conjugated alongwith the P54AR4-83v2 parent (SEQ ID NOS: 217 and 255 and Tencon (SEQ IDNO: 265) as a negative control.

Cell killing was assessed by measuring viability of theEGFR-overexpressing human tumor cell line H1573 following exposure tothe cysteine variant-cytotoxin conjugates. Cells were plated inblack-well, clear bottomed, tissue culture-treated plates (Falcon353219) at 7000/well in 100 μL/well of phenol red free RPMI media (Gibco11835-030) with 5% fetal bovine serum (Gibco). Cells were allowed toattach overnight at 37° C. in a humidified 5% CO₂ atmosphere. Medium wasaspirated from 96-well plate and cells were treated with 50 uL of freshmedia and 50 uL of 2× inhibitor made up in fresh media. Cell viabilitywas determined by an endpoint assay with Cell TiterGlo (Promega) at 70hours. IC₅₀ values were determined by fitting data to the equation for asigmoidal dose response with variable slope using GraphPad Prism 5(GraphPad Software). Table 11 reports IC₅₀ values obtained from analysisof the CellTiter Glo data. The average IC₅₀ of two replicates of the83v2-cys/vcMMAF conjugate was 0.7 nM. Four of the 32 conjugates testedhad IC₅₀ values more than two times that of the parent (above 1.4 nM)and were discarded: L32C, T68C, Y72C, and V74C. Additionally, threeconjugates gave IC₅₀ values over two times more potent than the parentand may be especially suitable for formatting into drug conjugates: N6C,E53C, and T93C.

TABLE 11 Variant IC50 (nM) N6C 0.16 V8C 0.35 S10C 0.43 E11C 0.94 E14C0.34 D15C 0.33 S16C 0.75 S20C 0.36 S30C 0.78 L32C 2.92 Q34C 0.74 S38C0.76 K40C 0.73 V41C 1.13 I45C 0.63 L47C 1.03 T48C 0.59 E53C 0.09 R54C0.37 T59C 0.44 G60C 1.00 K62C 1.25 G64C 0.36 T68C 3.70 S70C 1.14 Y72C1.85 V74C 3.13 L88C 0.81 S89C 0.94 A90C 1.00 I91C 0.54 T93C 0.2083v2His₆-cys (SEQ ID 0.61 NOs: 217 and 255) 83v2His₆-cys (SEQ ID 0.79NOs: 217 and 255) WT 146.00 WT 166.30Final Cysteine Variants

Of the 96 positions tested, 28 of the cysteine variants were found tomeet the criteria of retention of high expression level in E. coli,efficient conjugation via thiol-maleimide chemistry, retention ofbinding to target antigen EGFR, retention of thermostability andreversible unfolding properties, and retention of killing of cells withhigh EGFR expression when the cysteine variant is conjugated to acytotoxic drug. These positions are: N6C (SEQ ID NOS: 210 and 248), V8C(SEQ ID NOS: 189 and 227), S10C (SEQ ID NOS: 190 and 228), E11C (SEQ IDNOS: 191 and 229), E14C (SEQ ID NOS: 192 and 230), D15C (SEQ ID NOS: 193and 231), S16C (SEQ ID NOS: 194 and 232), S20C (SEQ ID NOS: 195 and233), S30C (SEQ ID NOS: 196 and 234), Q34C (SEQ ID NOS: 197 and 235),S38C (SEQ ID NOS: 198 and 236), K40C (SEQ ID NOS: 199 and 237), V41C(SEQ ID NOS: 200 and 238), I45C (SEQ ID NOS: 201 and 239), L47C (SEQ IDNOS: 202 and 240), T48C (SEQ ID NOS: 203 and 241), E53C (SEQ ID NOS: 204and 242), R54C (SEQ ID NOS: 205 and 243), T59C (SEQ ID NOS: 206 and244), G60C (SEQ ID NOS: 207 and 245), K62C (SEQ ID S 208 and 246), G64C(SEQ ID NOS: 209 and 247), T68C (SEQ ID NOS: 210 and 248), S70C (SEQ IDNOS: 211 and 249), L88C (SEQ ID NOS: 212 and 250), S89C (SEQ ID NOS: 213and 251), A90C (SEQ ID NOS: 214 and 252), 191C (SEQ ID NOS: 215 and253), and T93C (SEQ ID NOS: 216 and 254). The locations of these 28positions within the structure of the 83v2 protein are shown in FIG. 3.

Example 6: Selection of Fibronectin Type III (FN3) Domains that Bindc-Met and Inhibit HGF Binding

Panning on Human c-Met

The TCL14 library was screened against biotinylated-human c-Metextracellular domain (bt-c-Met) to identify FN3 domains capable ofspecifically binding c-Met. For selections, 3 μg of TCL14 library was invitro transcribed and translated (IVTT) in E. Coli S30 Linear Extract(Promega, Madison, Wis.) and the expressed library blocked with CisBlock (2% BSA (Sigma-Aldrich, St. Louis, Mo.), 100 μg/ml Herring SpermDNA (Promega), 1 mg/mL heparin (Sigma-Aldrich)). For selections,bt-c-Met was added at concentrations of 400 nM (Round 1), 200 nM (Rounds2 and 3) and 100 nM (Rounds 4 and 5). Bound library members wererecovered using neutravidin magnetic beads (Thermo Fisher, Rockford,Ill.) (Rounds 1, 3, and 5) or streptavidin magnetic beads (Promega)(Rounds 2 and 4) and unbound library members were removed by washing thebeads 5-14 times with 500 uL PBS-T followed by 2 washes with 500 μL PBS.

Additional selection rounds were performed to identify FN3 domainsmolecules with improved affinities. Briefly, outputs from round 5 wereprepared as described above and subjected to additional iterative roundsof selection with the following changes: incubation with bt-c-Met wasdecreased from 1 hour to 15 minutes and bead capture was decreased from20 minutes to 15 minutes, bt-c-Met decreased to 25 nM (Rounds 6 and 7)or 2.5 nM (Rounds 8 and 9), and an additional 1 hour wash was performedin the presence of an excess of non-biotinylated c-Met. The goal ofthese changes was to simultaneously select for binders with apotentially faster on-rate and a slower off-rate yielding asubstantially lower K_(D).

Rounds 5, 7 and 9 outputs were PCR cloned into a modified pET15 vector(EMD Biosciences, Gibbstown, N.J.) containing a ligase independentcloning site (pET15-LIC) using TCON6 (SEQID No. 30) and TCON5 E86I short(SEQID No. 31) primers, and the proteins were expressed as C-terminalHis6-tagged proteins after transformations and IPTG induction (1 mMfinal, 30° C. for 16 hours) using standard protocols. The cells wereharvested by centrifugation and subsequently lysed with Bugbuster HT(EMD Biosciences) supplemented with 0.2 mg/mL Chicken Egg White Lysozyme(Sigma-Aldrich). The bacterial lysates were clarified by centrifugationand the supernatants were transferred to new 96 deep-well plates.

Screening for FN3 Domains that Inhibit HGF Binding to c-Met

FN3 domains present in E. coli lysates were screened for their abilityto inhibit HGF binding to purified c-Met extracellular domain in abiochemical format. Recombinant human c-Met Fc chimera (0.5 μg/mL inPBS, 100 μL/well) was coated on 96-well White Maxisorp Plates (Nunc) andincubated overnight at 4° C. The plates were washed two times with 300μl/well of Tris-buffered saline with 0.05% Tween 20 (TBS-T,Sigma-Aldrich) on a Biotek plate washer. Assay plates were blocked withStartingBlock T20 (200 μL/well, Thermo Fisher Scientific, Rockland,Ill.) for 1 hour at room temperature (RT) with shaking and again washedtwice with 300 μl of TBS-T. FN3 domain lysates were diluted inStartingBlock T20 (from 1:10 to 1:100,000) using the Hamilton STARplusrobotics system. Lysates (50 μL/well) were incubated on assay plates for1 hour at RT with shaking. Without washing the plates, bt-HGF (1 μg/mLin StartingBlock T20, 50 μL/well, biotinylated) was added to the platefor 30 min at RT while shaking. Control wells containing Tencon27lysates received either Starting Block T20 or diluted bt-HGF. Plateswere then washed four times with 300 μl/well of TBS-T and incubated with100 μl/well of Streptavidin-HRP (1:2000 in TBS-T, JacksonImmunoresearch, West Grove, Pa.) for 30-40 minutes at RT with shaking.Again the plates were washed four times with TBS-T. To develop signal,POD Chemiluminescence Substrate (50 μL/well, Roche Diagnostics,Indianapolis, Ind.), prepared according to manufacturer's instructions,was added to the plate and within approximately 3 minutes luminescencewas read on the Molecular Devices M5 using SoftMax Pro. Percentinhibition was determined using the following calculation:100−((RLU_(sample)−Mean RLU_(No bt-HGF control))/(MeanRLU_(bt-HGF control)−Mean RLU_(No bt-HGF control))*100). Percentinhibition values of 50% or greater were considered hits.

High-Throughput Expression and Purification of FN3 Domains

His-tagged FN3 domains were purified from clarified E. coli lysates withHis MultiTrap™ HP plates (GE Healthcare) and eluted in buffer containing20 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole atpH 7.4. Purified samples were exchanged into PBS pH 7.4 for analysisusing PD MultiTrap™ G-25 plates (GE Healthcare).

IC₅₀ Determination of Inhibition of HGF Binding to c-Met

Select FN3 domains were further characterized in the HGF competitionassay. Dose response curves for purified FN3 domains were generatedutilizing the assay described above (starting concentrations of 5 μM).Percent inhibition values were calculated. The data were plotted as %inhibition against the logarithm of FN3 domain molar concentrations andIC₅₀ values were determined by fitting data to a sigmoidal dose responsewith variable slope using GraphPad Prism 4.

35 unique sequences were identified from Round 5 to exhibit activity atdilutions of 1:10, with IC₅₀ values ranging from 0.5 to 1500 nM. Round 7yielded 39 unique sequences with activity at dilutions of 1:100 and IC₅₀values ranging from 0.16 to 2.9 nM. 66 unique sequences were identifiedfrom Round 9, where hits were defined as being active at dilutions of1:1000. IC₅₀ values as low as 0.2 nM were observed in Round 9 (Table13).

Example 7: Characterization of FN3 Domains that Bind c-Met and InhibitHGF Binding

FN3 domains were expressed and purified as described above in Example 2.Size exclusion chromatography and kinetic analysis was done as describedabove in Examples 1 and 2, respectively. Table 12 shows the sequences ofthe C-strand, CD loop, F-strand, and FG loop, and a SEQ ID NO: for theentire amino acid sequence for each domain.

TABLE 12 Clone SEQ ID Name NO: C loop CD strand F loop FG strandP114AR5P74-A5 32 FDSFWIRYDE VVVGGE TEYYVNILGV KGGSISV P114AR5P75-E9 33FDSFFIRYDE FLRSGE TEYWVTILGV KGGLVST P114AR7P92-F3 34 FDSFWIRYFE FLGSGETEYIVNIMGV KGGSISH P114AR7P92-F6 35 FDSFWIRYFE FLGSGE TEYVVNILGV KGGGLSVP114AR7P92-G8 36 FDSFVIRYFE FLGSGE TEYVVQILGV KGGYISI P114AR7P92-H5 37FDSFWIRYLE FLLGGE TEYVVQIMGV KGGTVSP P114AR7P93-D11 38 FDSFWIRYFE FLGSGETEYVVGINGV KGGYISY P114AR7P93-G8 39 FDSFWIRYFE FLGSGE TEYGVTINGV KGGRVSTP114AR7P93-H9 40 FDSFWIRYFE FLGSGE TEYVVQIIGV KGGHISL P114AR7P94-A3 41FDSFWIRYFE FLGSGE TEYVVNIMGV KGGKISP P114AR7P94-E5 42 FDSFWIRYFE FLGSGETEYAVNIMGV KGGRVSV P114AR7P95-B9 43 FDSFWIRYFE FLGSGE TEYVVQILGV KGGSISVP114AR7P95-D3 44 FDSFWIRYFE FLGSGE TEYVVNIMGV KGGSISY P114AR7P95-D4 45FDSFWIRYFE FLGSGE TEYVVQILGV KGGYISI P114AR7P95-E3 46 FDSFWIRYFE FLGSGETEYVVQIMGV KGGTVSP P114AR7P95-F10 47 FDSFWIRYFE FTTAGE TEYVVNIMGVKGGSISP P114AR7P95-G7 48 FDSFWIRYFE LLSTGE TEYVVNIMGV KGGSISPP114AR7P95-H8 49 FDSFWIRYFE FVSKGE TEYVVNIMGV KGGSISPC loop residues correspond to residues 28-37 of indicated SEQ ID NO:CD strand residues correspond to residues 38-43 of indicated SEQ ID NO:F loop residues correspond to residues 65-74 of indicated SEQ ID NO:FG strand residues correspond to residues 75-81 of indicated SEQ ID NO:Binding of Selected c-Met-Binding FN3 Domains to c-Met on Cells

NCI-H441 cells (Cat # HTB-174, American Type Culture Collection,Manassas, Va.) were plated at 20,000 cells per well in Poly-D-lysinecoated black clear bottom 96-well plates (BD Biosciences, San Jose,Calif.) and allowed to attach overnight at 37° C., 5% CO₂. Purified FN3domains (50 μL/well; 0 to 1000 nM) were added to the cells for 1 hour at4° C. in duplicate plates. Supernatant was removed and cells were washedthree times with FACS stain buffer (150 μL/well, BD Biosciences, cat#554657). Cells were incubated with biotinylated-anti HIS antibody(diluted 1:160 in FACS stain buffer, 50 μL/well, R&D Systems, cat #BAM050) for 30 minutes at 4° C. Cells were washed three times with FACSstain buffer (150 μL/well), after which wells were incubated with antimouse IgG1-Alexa 488 conjugated antibody (diluted 1:80 in FACS stainbuffer, 50 μL/well, Life Technologies, cat # A21121) for 30 minutes at4° C. Cells were washed three times with FACS stain buffer (150 μL/well)and left in FACS stain buffer (50 μL/well). Total fluorescence wasdetermined using an Acumen eX3 reader. Data were plotted as rawfluorescence signal against the logarithm of the FN3 domain molarconcentration and fitted to a sigmoidal dose-response curve withvariable slope using GraphPad Prism 4 (GraphPad Software) to calculateEC₅₀ values. FN3 domains were found to exhibit a range of bindingactivities, with EC₅₀ values between 1.4 and 22.0, as shown in Table 13.

Inhibition of HGF-Stimulated c-Met Phosphorylation

Purified FN3 domains were tested for their ability to inhibitHGF-stimulated phosphorylation of c-Met in NCI-H441, using the c-Metphospho(Tyr1349) kit from Meso Scale Discovery (Gaithersburg, Md.).Cells were plated at 20,000/well in clear 96-well tissue culture-treatedplates in 100 μL/well of RPMI medium (containing Glutamax and HEPES,Life Technologies) with 10% fetal bovine serum (FBS; Life Technologies)and allowed to attach overnight at 37° C., 5% CO₂. Culture medium wasremoved completely and cells were starved overnight in serum-free RPMImedium (100 μL/well) at 37° C., 5% CO₂. Cells were then replenished withfresh serum-free RPMI medium (100 μL/well) containing FN3 domains at aconcentration of 20 μM and below for 1 hour at 37° C., 5% CO₂. Controlswere treated with medium only. Cells were stimulated with 100 ng/mLrecombinant human HGF (100 μL/well, R&D Systems cat #294-HGN) andincubated at 37° C., 5% CO₂ for 15 minutes. One set of control wells wasleft un-stimulated as negative controls. Medium was then completelyremoved and cells were lysed with Complete Lysis Buffer (50 μL/well,Meso Scale Discovery) for 10 minutes at RT with shaking, as permanufacturer's instructions. Assay plates configured for measuringphosphorylated c-Met were blocked with the provided blocking solution asper the manufacturer's instructions at room temperature for 1 hour.Plates were then washed three times with Tris Wash Buffer (200 μL/well,Meso Scale Discovery). Cell lysates (30 μL/well) were transferred toassay plates, and incubated at RT with shaking for 1 hour. Assay plateswere then washed four times with Tris Wash Buffer, after which ice-coldDetection Antibody Solution (25 μL/well, Meso Scale Discovery) was addedto each well for 1 hr at RT with shaking. Plates were again rinsed fourtimes with Tris Wash Buffer. Signals were detected by addition of 150Read Buffer (150 μL/well, Meso Scale Discovery) and reading on a SECTOR®Imager 6000 instrument (Meso Scale Discovery) usingmanufacturer-installed assay-specific default settings. Data wereplotted as electrochemiluminescence signal against the logarithm of FN3domain molar concentration and IC₅₀ values were determined by fittingdata to a sigmoidal dose response with variable slope using GraphPadPrism 4. FN3 domains were found to inhibit phosphorylated c-Met withIC50 values ranging from 4.6 to 1415 nM as shown in Table 13.

Inhibition of Human Tumor Cell Growth

Inhibition of c-Met-dependent cell growth was assessed by measuringviability of U87-MG cells (American Type Culture Collection, cat #HTB-14), following exposure to c-Met-binding FN3 domains. Cells wereplated at 8000 cells per well in opaque white 96-well tissueculture-treated plates (Nunc) in 100 μL/well of RPMI medium,supplemented with 10% FBS and allowed to attach overnight at 37° C., 5%CO₂. Twenty-four hours after plating, medium was aspirated and cellswere replenished with serum-free RPMI medium.

Twenty-four hours after serum starvation, cells were treated by additionof serum-free medium containing c-Met-binding FN3 domains (30 μL/well).Cells were incubated at 37° C., 5% CO₂ for 72 hours. Viable cells weredetected by addition of 100 μL/well of CellTiter-Glo® reagent (Promega),followed by mixing on a plate shaker for 10 minutes. Plates were read ona SpectraMax M5 plate reader (Molecular Devices) set to luminescencemode, with a read time of 0.5 seconds/well. Data were plotted as rawluminescence units (RLU) against the logarithm of FN3 domain molarconcentration. IC₅₀ values were determined by fitting data to anequation for a sigmoidal dose response with variable slope usingGraphPad Prism 4. Table 13 reports IC₅₀ values ranging from 1 nMto >1000 nM.

TABLE 13 Summary of biological properties of c-Met-binding FN3 domains.pMet Inhbibition of HGF H441 Cell inhibition in Proliferation of CloneAffinity competition binding H441 cells U87-MG cells Name SEQ ID NO:(Kd, nM) IC50 (nM) (EC50, nM) (IC50, nM) (IC50, nM) P114AR5P74-A5 3210.1 5.2 18.7 1078 464.4 P114AR5P75-E9 33 45.8 51.9 ND 1415 1193.9P114AR7P92-F3 34 0.4 0.2 1.5 8.3 2.7 P114AR7P92-F6 35 3.1 2.2 4.9 165.3350.5 P114AR7P92-G8 36 1.0 1.6 5.9 155.3 123.9 P114AR7P92-H5 37 11.6 ND22.0 766.4 672.3 P114AR7P93-D11 38 ND ND 2.3 16 14.4 P114AR7P93-G8 396.9 1 3.8 459.5 103.5 P114AR7P93-H9 40 3.3 2.9 12.9 288.2 269.9P114AR7P94-A3 41 0.4 0.2 1.4 5 9.3 P114AR7P94-E5 42 4.2 0.7 3.4 124.3195.6 P114AR7P95-B9 43 0.5 0.3 ND 9.8 17.4 P114AR7P95-D3 44 0.3 0.2 1.54.6 1.7 P114AR7P95-D4 45 0.4 ND 1.4 19.5 19.4 P114AR7P95-E3 46 1.5 ND3.2 204.6 209.2 P114AR7P95-F10 47 4.2 1.4 4.4 187.6 129.7 P114AR7P95-G748 20.0 ND 11.3 659.3 692 P114AR7P95-H8 49 3.7 ND 4.1 209.8 280.7Thermal Stability of c-Met-Binding FN3 DomainsDifferential scanning calorimetry in PBS was used to assess thestability of each FN3 domain. Results of the experiment are shown inTable 14.

TABLE 14 Thermal Clone Stability Name SEQ ID NO: (Tm, C.) P114AR5P74-A532 74.1 P114AR5P75-E9 33 ND P114AR7P92-F3 34 81.5 P114AR7P92-F6 35 76.8P114AR7P92-G8 36 90.9 P114AR7P92-H5 37 87 P114AR7P93-D11 38 NDP114AR7P93-G8 39 76.8 P114AR7P93-H9 40 88.2 P114AR7P94-A3 41 86.2P114AR7P94-E5 42 80 P114AR7P95-B9 43 86.3 P114AR7P95-D3 44 82P114AR7P95-D4 45 85.3 P114AR7P95-E3 46 94.2 P114AR7P95-F10 47 85.2P114AR7P95-G7 48 87.2 P114AR7P95-H8 49 83

Example 8. Generation and Characterization of Bispecific Anti-EGFR/c-MetMolecules

Generation of Bispecific EGFR/c-Met Molecules

Numerous combinations of the EGFR and c-Met-binding FN3 domainsdescribed in Examples 1-6 were joined into bispecific molecules capableof binding to both EGFR and c-Met. Additionally, EGFR-binding FN3domains having amino acid sequences shown in SEQ ID NOs: 107-110 andc-Met binding FN3 domains having amino acid sequences shown in SEQ IDNOs: 111-114 were made and joined into bispecific molecules. Syntheticgenes were created to encode for the amino acid sequences described inSEQID No. 50-72 and 106 (Table 15) such that the following format wasmaintained: EGFR-binding FN3 domain followed by a peptide linkerfollowed by a c-Met-binding FN3 domain. A poly-histidine tag wasincorporated at the C-terminus to aid purification. In addition to thosemolecules described in Table 15, the linker between the two FN3 domainswas varied according to length, sequence composition and structureaccording to those listed in Table 16. It is envisioned that a number ofother linkers could be used to link such FN3 domains BispecificEGFR/c-Met molecules were expressed and purified from E. coli asdescribed for monospecific EGFR or c-Met FN3 domains using IMAC and gelfiltration chromatography steps.

TABLE 15 Bispecifcic EGFR/c- EGFR-binding Met molecule FN3 comaincMET-binding FN3 domain Linker Clone ID SEQ ID Clone ID SEQ ID Clone IDSEQ ID Sequence SEQ ID ECB1 50 P54AR4-83v2 27 P114AR5P74-A5 32 (GGGGS)₄79 ECB2 51 P54AR4-83v2 27 P114AR7P94-A3 41 (GGGGS)₄ 79 ECB3 52P54AR4-83v2 27 P114AR7P93-H9 40 (GGGGS)₄ 79 ECB4 53 P54AR4-83v2 27P114AR5P75-E9 33 (GGGGS)₄ 79 ECB5 54 P53A1R5-17v2 107 P114AR7P94-A3 41(GGGGS)₄ 79 ECB6 55 P53A1R5-17v2 107 P114AR7P93-H9 40 (GGGGS)₄ 79 ECB756 P53A1R5-17v2 107 P114AR5P75-E9 33 (GGGGS)₄ 79 ECB15 57 P54AR4-83v2 27P114AR7P94-A3 41 (AP)₅ 81 ECB27 58 P54AR4-83v2 27 P114AR5P74-A5 32 (AP)₅81 ECB60 59 P53A1R5-17v2 107 P114AR7P94-A3 41 (AP)₅ 81 ECB37 60P53A1R5-17v2 107 P114AR5P74-A5 32 (AP)₅ 81 ECB94 61 P54AR4-83v22 108P114AR7P94-A3v22 111 (AP)₅ 81 ECB95 62 P54AR4-83v22 108 P114AR9P121-A6v2112 (AP)₅ 81 ECB96 63 P54AR4-83v22 108 P114AR9P122-A7v2 113 (AP)₅ 81ECB97 64 P54AR4-83v22 108 P114AR7P95-C5v2 114 (AP)₅ 81 ECB106 65P54AR4-83v23 109 P114AR7P94-A3v22 111 (AP)₅ 81 ECB107 66 P54AR4-83v23109 P114AR9P121-A6v2 112 (AP)₅ 81 ECB108 67 P54AR4-83v23 109P114AR9P122-A7v2 113 (AP)₅ 81 ECB109 68 P54AR4-83v23 109 P114AR7P95-C5v2114 (AP)₅ 81 ECB118 69 P53A1R5-17v22 110 P114AR7P94-A3v22 111 (AP)₅ 81ECB119 70 P53A1R5-17v22 110 P114AR9P121-A6v2 112 (AP)₅ 81 ECB120 71P53A1R5-17v22 110 P114AR9P122-A7v2 113 (AP)₅ 81 ECB121 72 P53A1R5-17v22110 P114AR7P95-C5v2 114 (AP)₅ 81 ECB91 106 P54AR4-83v22 108P114AR7P95-C5v2 114 (AP)₅ 81 ECB18 118 P54AR4-83v2 27 P114AR5P74-A5 32(AP)₅ 81 ECB28 119 P53A1R5-17v2 107 P114AR5P74-A5 32 (AP)₅ 81 ECB38 120P54AR4-83v2 27 P114AR7P94-A3 41 (AP)₅ 81 ECB39 121 P53A1R5-17v2 107P114AR7P94-A3 41 (AP)₅ 81

TABLE 16 Linker SEQ ength in ID amino Linker NO: acids Structure GS 78 2Disordered GGGGS 105 5 Disordered (GGGGS)₄ 79 20 Disordered (AP)₂ 80 4Rigid (AP)₅ 81 5 Rigid (AP)₁₀ 82 20 Rigid (AP)₂₀ 83 40 RigidA(EAAAK)₅AAA 84 29 α-helicalBispecific EGFR/c-Met Molecules Enhance Potency Compared to MonospecificMolecules Alone, Suggesting Avidity

NCI-H292 cells were plated in 96 well plates in RPMI medium containing10% FBS. 24 hours later, medium was replaced with serum free RPMI. 24hours after serum starvation, cells were treated with varyingconcentrations of FN3 domains: either a high affinity monospecific EGFRFN3 domain (P54AR4-83v2), a weak affinity monospecific c-Met FN3 domain(P114AR5P74-A5), the mixture of the two monospecific EGFR and c-Met FN3domains, or a bispecific EGFR/c-Met molecules comprised of the lowaffinity c-Met FN3 domain linked to the high affinity EGFR FN3 domain(ECB1). Cells were treated for 1 h with the monospecific or bispecificmolecules and then stimulated with EGF, HGF, or a combination of EGF andHGF for 15 minutes at 37° C., 5% CO₂. Cells were lysed with MSD LysisBuffer and cell signaling was assessed using appropriate MSD Assayplates, according to manufacturer's instructions, as described above.

The low affinity c-Met FN3 domain inhibited phosphorylation of c-Metwith an IC₅₀ of 610 nM (FIG. 6). As expected the EGFR FN3 domain was notable to inhibit c-Met phosphorylation and the mixture of themono-specific molecules looked identical to the c-Met FN3 domain alone.However, the bi-specific EGFR/c-Met molecule inhibited phosphorylationof c-Met with an IC₅₀ of 1 nM (FIG. 6), providing more than a 2-logshift in improving potency relative to the c-Met monospecific alone.

The potential for the bispecific EGFR/c-Met molecule to enhance theinhibition of c-Met and/or EGFR phosphorylation through an avidityeffect was evaluated in multiple cell types with variable c-Met and EGFRdensities and ratios (FIG. 7). NCI-H292, NCI-H441, or NCI-H596 cellswere plated in 96 well plates in RPMI medium containing 10% FBS. 24hours later, medium was replaced with serum free RPMI. 24 hours afterserum starvation, cells were treated with varying concentrations ofeither monospecific EGFR-binding FN3 domain, monospecific c-Met FN3domain, or a bispecific EGFR/c-Met molecule (ECB5, comprised ofP53A1R5-17v2 and P114AR7P94-A3). Cells were treated for 1 h with themonospecific or bispecific molecules and then stimulated with EGF, HGF,or a combination of EGF and HGF for 15 minutes at 37° C., 5% CO₂. Cellswere lysed with MSD Lysis Buffer and cell signaling was assessed usingappropriate MSD Assay plates, according to manufacturer's instructions,as described above.

FIG. 7 (A-C) shows the inhibition of EGFR using a monospecificEGFR-binding FN3 domain compared to a bispecific EGFR/cMet molecule inthree different cell lines. To assess avidity in an EGFR phosphorylationassay, a medium affinity EGFR-binding FN3 domain (1.9 nM) (P53A1R5-17v2)was compared to a bispecific EGFR/c-Met molecule containing the sameEGFR-binding FN3 domain linked to a high-affinity c-Met-binding FN3domain (0.4 nM) (P114AR7P94-A3). In H292 and H596 cells, inhibition ofphosphorylation of EGFR was comparable for the monospecific andbispecific molecules (FIGS. 7A and 7B), likely because these cell lineshave a high ratio of EGFR to c-Met receptors. To test this theory,inhibition of EGFR phosphorylation was evaluated in NCI-H441 cells whichexhibit more c-Met receptors than EGFR. Treatment of NCI-H441 cells withthe bispecific EGFR/c-Met molecule decreased the IC₅₀ for inhibition ofEGFR phosphorylation compared to the monospecific EGFR-binding FN3domain by 30-fold (FIG. 7C).

The potential for enhanced potency with a bi-specific EGFR/c-Metmolecule was evaluated in a c-Met phosphorylation assay using a moleculewith a high affinity to EGFR (0.26 nM) and medium affinity to c-Met(10.1 nM). In both NCI-H292 and NCI-H596 cells, the inhibition ofphosphorylation of c-Met was enhanced with the bispecific moleculecompared to the monospecific c-Met-binding FN3 domain, by 134 and 1012fold, respectively (FIGS. 7D and 7E).

It was verified that the enhanced potency for inhibition of EGFR andc-Met phosphorylation with the bispecific EGFR/c-Met moleculestranslated into an enhanced inhibition of signaling and proliferation.For these experiments, the mixture of FN3 EGFR-binding and c-Met-bindingFN3 domains was compared to a bispecific EGFR/c-Met molecule. Asdescribed in Tables 17 and 18, the IC₅₀ values for ERK phosphorylation(Table 17) and proliferation of H292 cells (Table 18) were decreasedwhen cells were treated with the bispecific EGFR/c-Met molecule comparedto the mixture of the monospecific binders. The IC₅₀ for inhibition ofERK phosphorylation for the bi-specific EGFR/c-Met molecule was 143-foldlower relative to the mixture of the two monospecific EGFR and c-Met FN3domains, showing the effect of avidity to the potency of the moleculesin this assay. In Table 17, the monospecific EGFR- and c-Met binding FN3domains do not fully inhibit activity and therefore the IC₅₀ valuesshown should be considered lower limits. The proliferation assay wascompleted using different combinations EGFR and c-Met binding FN3domains either as a mixture or linked in a bispecific format. The IC₅₀for inhibition of proliferation for the bispecific EGFR/c-Met moleculewas 34-236-fold lower relative to the mixture of the monospecific parentEGFR or c-Met binding FN3 domains. This confirmed that the avidityeffect observed at the level of the receptors (FIG. 6 and FIG. 7)translates into an improvement in inhibiting cell signaling (Table 17)and cell proliferation (Table 18).

TABLE 17 Specificity of the FN3-domain IC50 (nM) (ERK molecule Clone #Type phosphorylation) EGFR P54AR4-83v2 monospecific >10,000 c-MetP114AR5P74-A5 monospecific 2366 EGFR or c-Met P54AR4-83v2 +P114AR5P74-A5 mixture of 798.4 monospecific molecules EGFR and c-MetECB1 bispecific 5.6

TABLE 18 Fold IC50 increase for in IC50 mixture IC50 for of forbispecific/ EGFR-binding c-Met binding mono- bi- mixture of FN3 domainFN3 domain specifics specific mono- (affinity) (affinity) (nM) (nM)specifics P54AR4-83v2 P114ARP94-A3 36.5 1.04 35 (0.26 nM) (0.4 nM)P54AR4-83v2 P114AR7P93-H9 274.5 8.05 34 (0.26 nM) (3.3 nM) P54AR4-83v2P114AR5P74-A5 1719 7.29 236 (0.26 nM) (10.1 nM)In Vive Tumor Xenografts: PK/PD

In order to determine efficacy of the monospecific and bispecific FN3domain molecules in vivo, tumor cells were engineered to secrete humanHGF (murine HGF does not bind to human HGF). Human HGF was stablyexpressed in NCI-H292 cells using lentiviral infection (Lentiviral DNAvector expressing human HGF (Accession # X16322) and lentiviralpackaging kit from Genecopoeia). After infection, HGF-expressing cellswere selected with 4 μg/mL puromycin (Invitrogen). Human HGF protein wasdetected in the conditioned medium of pooled cells using assay platesfrom MesoScale Discovery.

SCID Beige mice were subcutaneously inoculated with NCI-H292 cellsexpressing human HGF (2.0×10⁶ cells in Cultrex (Trevigen) in a volume of200 μL) on the dorsal flank of each animal. Tumor measurements weretaken twice weekly until tumor volumes ranged between 150-250 mm³. Micewere then given a single IP dose of bispecific EGFR/c-Met molecules(linked to an albumin binding domain to increase half-life) or PBSvehicle. At 6 h or 72 h after dosing, tumors were extracted andimmediately frozen in liquid nitrogen. Blood samples were collected viacardiac puncture into 3.8% citrate containing protease inhibitors.Immediately after collection, the blood samples were centrifuged and theresulting plasma was transferred to sample tubes and stored at −80° C.Tumors were weighed, cut into small pieces, and lysed in Lysing Matrix Atubes (LMA) containing RIPA buffer with HALT protease/phosphataseinhibitors (Pierce), 50 mM sodium fluoride (Sigma), 2 mM activatedsodium orthovanadate (Sigma), and 1 mM PMSF (MesoScale Discovery).Lysates were removed from LMA matrix and centrifuged to remove insolubleprotein. The soluble tumor protein was quantified with a BCA proteinassay and diluted to equivalent protein levels in tumor lysis buffer.Phosphorylated c-Met, EGFR and ERK were measured using assay plates fromMesoScale Discovery (according to Manufacturer's protocol and asdescribed above).

FIG. 6 shows the results of the experiments. Each bispecific EGFR/c-Metmolecule significantly reduced the levels of phosphorylated c-Met, EGFR,and ERK at both 6 h and 72 h. The data presented in FIG. 6 show theimportance of inhibiting both c-Met and EGFR simultaneously and how theaffinity of the bispecific EGFR/c-Met molecule for each receptor plays arole in inhibition of downstream ERK. The molecules containing the highaffinity EGFR-binding FN3 domains (P54AR4-83v2; shown as “8” in theFigure, K_(D)=0.26 nM) inhibited phosphorylation of EGFR to a largerextent compared to those containing the medium affinity EGFR-binding FN3domains (P53A1R5-17v2; shown as “17” in the figure K_(D)=1.9 nM) at both6 h and 72 h. All four bispecific molecules tested completely inhibitedphosphorylation of ERK at the 6 hour time point, regardless of affinity.At the 72 hour time point, the molecules containing the tight affinityc-Met-binding domain (P114AR7P94-A3; shown as “A3” in the figureK_(D)=0.4 nM) significantly inhibited phosphorylation of ERK compared tothe medium affinity c-Met-binding FN3 domain (P114AR5P74-A5; shown as“A5” in the Figure; K_(D)=10.1 nM; FIG. 6).

The concentration of each bispecific EGFR/c-Met molecule was measured at6 and 72 hours after dosing in the blood and in the tumor (FIG. 9).Interestingly, the bispecific molecule with the medium affinityEGFR-binding domain (P53A1R5-17v2; K_(D)=1.9 nM) but high affinityc-Met-binding FN3 domain (P114AR7P94-A3; K_(D)=0.4 nM) had significantlymore tumor accumulation at 6 hours relative to the other molecules,while the difference is diminished by 72 hours. It can be hypothesizedthat cells outside the tumor have lower levels of both EGFR and c-Metsurface expression and therefore the medium affinity EGFR moleculedoesn't bind to normal tissue as tightly compared to the higher affinityEGFR-binding FN3 domain. Therefore there is more free medium affinityEGFR-binding FN3 domain available to bind in the tumor. Therefore,identifying the appropriate affinities to each receptor may allow foridentification of a therapeutic with decreased systemic toxicities andincreased tumor accumulation.

Tumor Efficacy Studies with Bispecific EGFR/c-Met Molecules

SCID Beige mice were subcutaneously inoculated with NCI-H292 cellsexpressing human HGF (2.0×10⁶ cells in Cultrex (Trevigen) in 200 μL) inthe dorsal flank of each animal. One week after implantation, mice werestratified into groups with equivalent tumor volumes (mean tumorvolume=77.9+/−1.7 mm³). Mice were dosed three times per week with thebispecific molecules and tumor volumes were recorded twice weekly. Tumorgrowth inhibition (TGI) was observed with four different bispecificmolecules, with variable affinities for c-Met and EGFR. FIG. 10 showsthe benefit of inhibiting both c-Met and EGFR as a delay in tumor growthwas observed in the mice treated with molecules containing the highaffinity EGFR-binding FN3 domain compared to the medium affinityEGFR-binding FN3 domain when the c-Met-binding FN3 domain was mediumaffinity (open vs. closed triangles, P54AR4-83v2-P114AR5P74-A5 comparedto P53A1R5-17-P114AR5P74-A5). In addition, the data shows the importanceof having a high affinity c-Met-binding FN3 domain as bispecificmolecules containing either the high or medium affinity EGFR-binding FN3domain but high affinity c-Met-binding FN3 domain showed the mostefficacy (dotted gray and black lines, P54AR4-83v2-P114AR7P94-A3 andP53A1R5-17v2-P114AR7P94-A3).

Efficacy of Bispecfic Molecule and Other Inhibitors of EGFR and c-Met

The in vivo therapeutic efficacies of a bispecific EGFR/c-Met molecule(ECB38) and the small molecule inhibitors crizotinib (c-Met inhibitor)and erlotinib (EGFR inhibitor), cetuximab (anti-EGFR antibody), each asa single agent, and the combination of crizotinib and erlotinib, wereevaluated in the treatment of subcutaneous H292-HGF human lung cancerxenograft model in SCID/Beige mice (FIG. 11).

The H292-HGF cells were maintained in vitro in RPMI1640 mediumsupplemented with fetal bovine serum (10% v/v), and L-glutamine (2 mM)at 37° C. in an atmosphere of 5% CO2 in air. The cells were routinelysubcultured twice weekly by trypsin-EDTA treatment. The cells growing inan exponential growth phase were harvested and counted for tumorinoculation.

Each mouse was inoculated subcutaneously at the right flank region withH292-HGF tumor cells (2×10⁶) in 0.1 ml of PBS with cultrex (1:1) fortumor development. The treatments were started when the mean tumor sizereached 139 mm³. The test article administration and the animal numbersin each study group were shown in the following experimental designtable (Table 26). The date of tumor cell inoculation was denoted as day0.

TABLE 26 Dose Planned Actual Group N Treatment (mg/kg) Dosing RouteSchedule Schedule 1 10 Vehicle 0 i.p. QD × 3 QD × 3 Control weeks weeks2 10 bispecific 25 i.p. 3 3 EGFR/c-Met times/week × times/week ×molecule 3 weeks 3 weeks 3 10 Crizotinib 50 p.o. QD × 3 QD × 17 daysweeks 4 10 Erlotinib 50 p.o. QD × 2 QD × 3 weeks weeks 5 10 Crizotinib50 p.o. QD × 3 QD × 3 weeks weeks Erlotinib 50 p.o. QD × 2 QD × 3 weeksweeks 6 10 Cetuximab 1 mg/mouse i.p. Q4d*6 Q4d*6 N: animal number; p.o.:oral administration; i.p.: intraperitoneal injection 3 times/week: doseswere given on days 1, 3 and 5 of the week. QD: once daily Q4d: onceevery four days; the interval of the combination of crizotinib anderlotinib was 0.5 hrs; dosing volume was adjusted based on body weight(10 l/g); a: dosing was not given on day 14 post grouping.

Before commencement of treatment, all animals were weighed and the tumorvolumes were measured. Since the tumor volume can affect theeffectiveness of any given treatment, mice were assigned into groupsusing randomized block design based upon their tumor volumes. Thisensures that all the groups are comparable at the baseline. Therandomized block design was used to assign experimental animals togroups. First, the experimental animals were divided into homogeneousblocks according to their initial tumor volume. Secondly, within eachblock, randomization of experimental animals to treatments wasconducted. Using randomized block design to assign experimental animalsensured that each animal had the same probability of being assigned to agiven treatment and therefore systematic error was reduced.

At the time of routine monitoring, the animals were checked for anyeffects of tumor growth and treatments on normal behavior, such asmobility, visual estimation of food and water consumption, body weightgain/loss (body weights were measured twice weekly), eye/hair mattingand any other abnormal effect.

The major endpoint was whether tumor growth can be delayed or tumorbearing mice can be cured. Tumor size was measured twice weekly in twodimensions using a caliper, and the volume was expressed in mm³ usingthe formula: V=0.5a×b² where a and b are the long and short diameters ofthe tumor, respectively. The tumor size was then used for calculationsof both T-C and T/C values. T-C was calculated with T as the time (indays) required for the mean tumor size of the treatment group to reach1000 mm³, and C was the time (in days) for the mean tumor size of thecontrol group to reach the same size. The T/C value (in percent) was anindication of antitumor efficacy; T and C were the mean volume of thetreated and control groups, respectively, on a given day. Complete tumorregression (CR) is defined as tumors that are reduced to below the limitof palpation (62.5 mm³). Partial tumor regression (PR) is defined astumors that are reduced from initial tumor volume. A minimum duration ofCR or PR in 3 or more successive tumor measurements is required for a CPor PR to be considered durable.

Animals for which the body weight loss exceeded 20%, or for which themean tumor size of the group exceeds 2000 mm³ were euthanized. The studywas terminated after two weeks of observation after the final dose.

Summary statistics, including mean and the standard error of the mean(SEM), are provided for the tumor volume of each group at each timepoint (shown in Table 19 below). Statistical analyses of difference intumor volume among the groups were evaluated using a one-way ANOVAfollowed by individual comparisons using Games-Howell (equal variancenot assumed). All data were analyzed using SPSS 18.0. p<0.05 wasconsidered to be statistically significant.

TABLE 19 Tumor Sizes in Treatment Groups Tumor volume (mm³)a bispecificEGFR/c- Crizotinib; Met Crizotinib Erlotinib at Cetuximab molecule at atErlotinib at 50 mg/kg; at 1 mg/ Days Vehicle 25 mg/kg 50 mg/kg 50 mg/kg50 mg/kg mouse 7 139 ± 7  137 ± 7  140 ± 9  141 ± 8  139 ± 8  139 ± 10 9230 ± 20 142 ± 7  217 ± 20 201 ± 19 134 ± 9  168 ± 13 13 516 ± 45 83 ± 6547 ± 43 392 ± 46 109 ± 10 212 ± 20 16  808 ± 104 44 ± 7 914 ± 92 560 ±70 127 ± 15 252 ± 28 20 1280 ± 209 30 ± 6 1438 ± 239  872 ± 136 214 ± 30371 ± 48 23 1758 ± 259 23 ± 7 2102 ± 298 1122 ± 202 265 ± 40 485 ± 61 272264 ± 318 21 ± 5 — 1419 ± 577 266 ± 42 640 ± 82 30 — 23 ± 6 — 1516 ±623 482 ± 61  869 ± 100

The mean tumor size of the vehicle treated group (Group 1) reached 1,758mm³ at day 23 after tumor inoculation. Treatment with the bispecificEGFR/c-Met molecule at 25 mg/kg dose level (Group 2) led to completetumor regression (CR) in all mice which were durable in >3 successivetumor measurements (average TV=23 mm³, T/C value=1%, p=0.004 comparedwith the vehicle group at day 23).

Treatment with Crizotinib as a single agent at 50 mg/kg dose level(Group 3) showed no antitumor activity; the mean tumor size was 2,102mm³ at day 23 (T/C value=120%, p=0.944 compared with the vehicle group).

Treatment with Erlotinib as a single agent at 50 mg/kg dosing level(Group 4) showed minor antitumor activity, but no significant differencewas found compared with the vehicle group; the mean tumor size was 1,122mm³ at day 23 (T/C value=64%, p=0.429 compared with the vehicle group),with 4 days of tumor growth delay at tumor size of 1,000 mm³ comparedwith the vehicle group.

The combination of Crizotinib (50 mg/kg, Group 5) and Erlotinib (50mg/kg, Group 5) showed significant antitumor activity; the mean tumorsize was 265 mm³ at day 23 (T/C=15%; p=0.008), with 17 days of tumorgrowth delay at tumor size of 1,000 mm³ compared with the vehicle group.

Cetuximab at 1 mg/mouse dosing level as a single agent (Group 6) showedsignificant antitumor activities; the mean tumor size was 485 mm³ at day23 (T/C=28%; p=0.018), with 17 days of tumor growth delay at tumor sizeof 1,000 mm³ compared with the vehicle group. FIG. 11 shows theanti-tumor activities of the various therapies.

TABLE 20 Anti-Tumor Activity T-C Tumor Size (days) at (mm³)a T/C 1000 PTreatment at day 23 (%) mm³) value Vehicle 1758 ± 259  — — — bispecific23 ± 7  1 — 0.004 EGFR/c-Met molecule (25 mg/kg) Crizotinib 2102 ± 298 120 −1 0.944 (50 mg/kg) Erlotinib 1122 ± 202  64 4 0.429 (50 mg/kg)Crizotinib + 265 ± 40  15 17 0.008 Erlotinib (50 mg/kg + 50 mg/kg)Cetuximab 485 ± 61  28 17 0.018 (1 mg/mouse)

Medium to severe body weight loss was observed in the vehicle groupwhich might be due to the increasing tumor burden; 3 mice died and 1mouse were euthanized when BWL>20% by day 23. Slight toxicity of thebispecific EGFR/c-Met molecule was observed in Group 2; 3 mice wereeuthanized when BWL>20% during the treatment period; the body weight wasgradually recovered when the treatment was withdrawn during the 2 weeksof observation period. More severe body weight loss was observed in theCrizotinib or Erlotinib monotherapy group compared to the vehicle group,suggesting the treatment related toxicity. The combination of Crizotiniband Erlotinib was generally tolerated during the dosing phase, butsevere body weight loss was observed at the end of the study, whichmight be due to the resumption of the fast tumor growth during thenon-treatment period. The monotherapy of Cetuximab was well tolerated inthe study; body weight loss was only observed at the end of the studydue to the resume of the tumor growth.

In summary, the bispecific EGFR/c-Met molecule at 25 mg/kg (3times/week×3 weeks) produced a complete response in H292-HGF human lungcancer xenograft model in SCID/Beige mice. The treatment was toleratedin 7 out of 10 mice, and resulted in severe body weight loss in 3 out of10 mice. FIG. 11 and Table 20 shows the impact of the various therapieson tumor size during the time points after treatment.

Example 9: Half-Life Extension of the Bispecific EGFR/c-Met Molecules

Numerous methods have been described to reduce kidney filtration andthus extend the serum half-life of proteins including modification withpolyethylene glycol (PEG) or other polymers, binding to albumin, fusionto protein domains which bind to albumin or other serum proteins,genetic fusion to albumin, fusion to IgG Fc domains, and fusion to long,unstructured amino acid sequences.

Bispecific EGFR/c-Met molecules were modified with PEG in order toincrease the hydrodynamic radius by incorporating a free cysteine at theC-terminus of the molecule. Most commonly, the free thiol group of thecysteine residue is used to attach PEG molecules that are functionalizedwith maleimide or iodoacetemide groups using standard methods. Variousforms of PEG can be used to modify the protein including linear PEG of1000, 2000, 5000, 10,000, 20,000, or 40,000 kDa. Branched PEG moleculesof these molecular weights can also be used for modification. PEG groupsmay also be attached through primary amines in the bispecific EGFR/c-Metmolecules in some instances.

In addition to PEGylation, the half-life of bispecific EGFR/c-Metmolecules was extended by producing these proteins as fusion moleculeswith a naturally occurring 3-helix bundle serum albumin binding domain(ABD) or a consensus albumin binding domain (ABDCon). These proteindomains were linked to the C-terminus of the c-Met-binding FN3 domainvia any of the linkers described in Table 16. The ABD or ABDCon domainmay also be placed between the EGFR-binding FN3 domain and the c-Metbinding FN3 domain in the primary sequence.

Example 10: Characterization of Select Bispecific EGFR/c-Met Molecules

Select EGFR/c-Met molecules were characterized for their affinity toboth EGFR and c-Met, their ability to inhibit EGFR and c-Metautophosphorylation, and their effect on proliferation of HGF cells.Binding affinity of the bispecific EGFR/c-Met molecules to recombinantEGFR and/or c-Met extracellular domain was further by surface Plasmonresonance methods using a Proteon Instrument (BioRad) according toprotocol described in Example 3. Results of the characterization areshown in Table 21.

TABLE 21 H292-HGF H292 Proliferation pMet pEGFR inhibition in K_(D)K_(D) inhibition inhibition in HGF-induced (EGFR, (c-Met, in H441 cellsH292 cells H292 cells nM) nM) (IC50, nM) (IC50, nM) (IC50, nM) ECB15 0.22.6 n/a 4.2 23 ECB94 1 4.3 53.8 5.1 29.6 ECB95 1.1 6.2 178.8 13.6 383.4ECB96 1.6 22.1 835.4 24.7 9480 ECB97 1.3 1.7 24.2 16.6 31.0 ECB106 16.75.1 53.3 367.4 484.5 ECB107 16.9 9 29.9 812.3 2637 ECB108 15.3 25.5126.2 814.4 11372 ECB109 17.3 2.1 26 432 573.6

Example 11: Generation and Characterization of Cysteine EngineeredBispecific Anti-EGFR/c-Met Molecules

Generation of Bispecific EGFR/c-Met Molecules

Based on the data generated from the cysteine scanning of theP54AR4-83v2 mutant (Example 5), cysteine mutants were also designed in abispecific anti-EGFR/c-Met molecule denoted ECB147 (SEQ ID NOS: 218 and256), which consists of the P54AR4-83v2 (SEQ ID NO: 27), the cMet binderP114AR7P95-C5v2 (SEQ ID NO: 114), and an albumin binding domain forhalf-life extension. These three domains are connected by (Ala-Pro)₅linkers (SEQ ID NO: 81). Variants with one, two, or four cysteines weredesigned with substitutions at the C-terminus, in the linker regions, orat the Lys-62 position of one of the FN3 domains (SEQ ID NOS: 219-225and 257-263). Another bispecific variant, ECB82cys (SEQ ID NOS: 226 and264) consists of P54AR4-83v2 (SEQ ID NO: 27), P114AR7P94-A3v22 (SEQ IDNO: 111), and a variant of the albumin-binding domain, all three domainsconnected by AP linkers, and a single C-terminal cysteine. An additionalcysteine variant of the non-targeted Tencon scaffold (SEQ ID NO: 265)was also used for the construction of control conjugates. All thevariants were constructed, expressed, and purified as described inprevious examples. Purity was assessed by SDS-PAGE analysis. Analyticalsize exclusion chromatography using a Superdex 75 5/150 column (GEHealthcare) shows that the FN3 domain preparations are free ofaggregates and elute at a time consistent with a monomeric protein. Massspectrometry determined the masses to be in agreement with thetheoretical masses (Table 22).

TABLE 22 Expected Experimental MW MW Variant Name (Da) (Da) ECB147v127895 27894 ECB147v2 27838 27837 ECB147v3 27877 27876 ECB147v4 2789527894 ECB147v5 27813 27812 ECB147v6 27838 27837 ECB147v7 27927 27926P54AR4-83v2-cys 11789 11790 Tencon-cys 10820Chemical Conjugation

To chemically conjugate the purified bispecific cysteine variants tomaleimide-containing molecules, the proteins were first reduced withTCEP to generate free thiols. 1-2 mg of each bispecific cysteine variantwas mixed with an excess of TCEP at neutral pH (Sigma catalog #646547)and incubated at RT for 30-60 minutes. TCEP was removed by adding 3volumes of saturated ammonium sulfate solution (4.02 M) to precipitatethe cysteine variants. After centrifugation at 16000-20000×g at 4° C.for 20 min and removal of the supernatant, the protein pellet wasdissolved in PBS or sodium phosphate buffer and mixed immediately with a5- to 10-fold excess of the maleimide-containing molecule. The reactionwas incubated for 30-60 minutes at room temperature and then quenchedwith an excess of a free thiol, such as cysteine or β-mercaptoethanol,to scavenge excess maleimide. The unbound maleimide was removed withZeba desalting columns (Thermo catalog #89890), by preparative SEC witha Tosoh G3000SW×1 column (# P4619-14N; 7.8 mm×30 cm; 5 Gun), or bybinding the cysteine variant to Ni-NTA resin, washing, and elutingessentially as described above. Conjugates were characterized bySDS-PAGE and mass spectrometry. This general method was used toconjugate bispecific cysteine variants to fluorescein maleimide (Thermocatalog #62245), PEG24-maleimide (Quanta Biodesign catalog #10319), andmaleimide-cytotoxin molecules with a variety of linkers (see structuresin FIG. 2).

Inhibition of EGF-Stimulated EGFR Phosphorylation

Purified bispecific PEG24-maleimide conjugates were tested for theirability to inhibit EGF-stimulated phosphorylation of EGFR in the humantumor cell line NCI-H292 (American Type Culture Collection, cat. #CRL-1848) using the EGFR phospho(Tyr1173) kit from Meso Scale Discovery(Gaithersburg, Md.) and as described in Example 3. The conjugates werecompared to unmodified ECB38 (SEQID No. 109), which differs from ECB147by two amino acids. The conjugates and ECB38 inhibited EGFR with similarIC₅₀ values, as shown in Table 23, demonstrating that modification atthe designed sites does not significantly affect target binding.

TABLE 23 Protein Name IC₅₀ (nM) ECB38 2.3 ECB147v3-PEG24 1.6ECB147v5-PEG24 0.9 ECB147v6-PEG24 1.4 ECB147v7-PEG24 1.4Inhibition of HGF-Stimulated c-Met Phosphorylation

Purified bispecific PEG24-maleimide conjugates were also tested fortheir ability to inhibit HGF-stimulated phosphorylation of c-Met inNCI-H292 cells, using the c-Met phosphor (Tyr1349) kit from Meso ScaleDiscovery (Gaithersburg, Md.), and as described in Example 7. Theconjugates and ECB38 inhibited cMet with similar IC₅₀ values as shown inTable 24, demonstrating that modification at these sites does notsignificantly alter target binding.

TABLE 24 Protein Name IC₅₀ (nM) ECB38 1.3 ECB147v3-PEG24 0.5ECB147v5-PEG24 0.4 ECB147v6-PEG24 0.4 ECB147v7-PEG24 0.5Cytotoxicity Assay

Conjugates consisting of ECB147 cysteine variants, 83v2-cys, orTencon-cys linked to a cytotoxic tubulin inhibitor from the auristatinfamily (FIG. 2) were tested for target-dependent cytotoxicity in cancercells. The inhibitor was linked to the cysteine-containing protein via anon-cleavable PEG₄ linker or an enzyme-cleavable valine-citrulline orvaline-lysine linker. Cell killing was assessed by measuring viabilityof the EGFR-overexpressing human tumor cell lines H1573 and A431 as wellas the EGFR-negative tumor cell line MDA-MB-435 following exposure tothe protein-cytotoxin conjugates using the procedure described inExample 4. Table 25 reports IC₅₀ values obtained from analysis of eitherthe CellTiter Glo or IncuCyte object count data at the 66, 72, or 90hour time point. The protein-drug conjugates showed potent cell-killingof cells that express the target antigen EGFR. The multi-drug conjugatesalso demonstrated increased cytotoxicity in many of the cell linestested.

TABLE 25 MMAE conjugates IC50 IC50 H1537 IC50 A431 MDA-MB-435 Conjugate(nM) (nM) (nM) TenconCys-mal-PEG4-MMAE ND >500TenconCys-mal-PEG4-VC-MMAE ND 841 poor fit TenconCys-mal-PEG4-VK-MMAE ND4.5 poor fit 83v2cy5-mal-PEG4-MMAE ND >500 83v2cy5-mal-PEG4-VC-MMAE ND315 512 83v2cy5-mal-PEG4-VK-MMAE ND 19.6 62 TenconCys-mal-PEG4-MMAFND >1000 TenconCys-mal-PEG4-VC-MMAF 996 >500 >500 1541TenconCys-mal-PEG4-VK-MMAF ND >500 >500 83v2cy5-mal-PEG4-MMAF ND >100083v2cy5-mal-PEG4-VC-MMAF 1 .19 1.6 >500 1.05 83v2cy5-mal-PEG4-VK-MMAF ND3.9 >500 ECB147v3-(mal-PEG4-VC-MMAF)₄ 0.15 0.0078 ND 0.075 0.0197ECB147v5-(mal-PEG4-VC-MMAF)₂ 0.056 0.087 ND 0.050 0.071ECB82cys-mal-PEG4-VC-MMAF 0.576 1.1 ND 0.249 0.64

SEQUENCE LISTING SEQ ID NO: Type Species Description Sequence 1 PRTArtificial Tencon LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT 2 DNA Artificial POP2220GGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGT TTCTGAAGTTACC 3 DNAArtificial TC5′toFG AACACCGTAGATAGAAACGGT 4 DNA Artificial 130merCGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTG 5 DNA Artificial POP2222CGGCGGTTAGAACGCGGCTAC 6 DNA Artificial TCF7GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNN AACACCGTAGATAGAAACGGT 7DNA Artificial TCF8 GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 8 DNA Artificial TCF9GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 9 DNA Artificial TCF10GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 10 DNA Artificial TCF11GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 11 DNA Artificial TCF12GGTGGTGAATTCCGCAGACAGCGGSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNSNNAACACCGTAGATAGAAACGGT 12 DNA Artificial POP2234AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCATGGTGATGGTGATGGTGACCGCCGGTGGTGAATTCCGCAGACAG 13 DNA Artificial POP2250CGGCGGTTAGAACGCGGCTACAATTAATAC 14 DNA Artificial DidLigRevCATGATTACGCCAAGCTCAGAA 15 DNA Artificial Tcon5new2GAGCCGCCGCCACCGGTTTAATGGTGATGGTGATGGT GACCACCGGTGGTGAATTCCGCAGACAG 16DNA Artificial Tcon6 AAGAAGGAGAACCGGTATGCTGCCGGCGCCGAAAAAC 17 DNAArtificial LS1008 TTTGGGAAGCTTCTAGGTCTCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCCCAAC TGATCTTCACCAAAC 18 PRT ArtificialP53A1R5- LPAPKNLVVSEVTEDSLRLSWADPHGFYDSFLIQYQES 17 withoutEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV met YKDTNMRGLPLSAEFTT 19 PRTArtificial P54AR4-17 LPAPKNLVVSEVTEDSLRLSVVTYDRDGYDSFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 20 PRTArtificial P54AR4-47 LPAPKNLVVSEVTEDSLRLSWGYNGDHFDSFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 21 PRTArtificial P54AR4-48 LPAPKNLVVSEVTEDSLRLSWDDPRGFYESFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 22 PRTArtificial P54AR4-37 LPAPKNLVVSEVTEDSLRLSWTWPYADLDSFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 23 PRTArtificial 54AR4-74 LPAPKNLVVSEVTEDSLRLSWGYNGDHFDSFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 24 PRTArtificial P54AR4-81 LPAPKNLVVSEVTEDSLRLSWDYDLGVYFDSFLIQYQE without metSEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHN VYKDTNMRGLPLSAEFTT 25 PRTArtificial P54AR4-83 LPAPKNLVVSEVTEDSLRLSWDDPWAFYESFLIQYQES without metEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 26 PRTArtificial P54CR4-31 LPAPKNLVVSEVTEDSLRLSVVTAPDAAFDSFLIQYQESE withoutMet KVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSY VFEHDVMLPLSAEFTT 27 PRTArtificial P54AR4-83v2 LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQES withoutMet EKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAIFTT 28 PRTArtificial P54CR4-31v2 LPAPKNLVVSEVTEDSARLSVVTAPDAAFDSFLIQYQESE withoutMet KVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSY VFEHDVMLPLSAIFTT 29 PRTArtificial P54AR4-73v2 LPAPKNLVVSEVTEDSLRLSWTWPYADLDSFLIQYQES wihtoutMet EKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNV YKDTNMRGLPLSAEFTT 30 DNAArtificial TCON6 AAG AAG GAG AAC CGG TAT GCT GCC GGC GCC GAA AAA C 31DNA Artificial TCON5 GAG CCG CCG CCA CCG GTT TAA TGG TGA TGG TGAE86Ishort TGG TGA CCA CCG GTG GTG AAG ATC GCA GAC AG 32 PRT ArtificialP114AR5P74- LPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYDEV A5VVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGG SISVPLSAIFTT 33 PRT ArtificialP114AR5P75- LPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFFIRYDEFL E9RSGEAIVLTVPGSERSYDLTGLKPGTEYVVVTILGVKGGL VSTPLSAIFTT 34 PRT ArtificialP114AR7P92- LPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYFEFL F3GSGEAIVLTVPGSERSYDLTGLKPGTEYIVNIMGVKGGSI SHPLSAIFTT 35 PRT ArtificialP114AR7P92- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL F6GSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGGL SVPLSAIFTT 36 PRT ArtificialP114AR7P92- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIRYFEFLG G8SGEAIVLTVPGSERSYDLTGLKPGTEYVVQILGVKGGYISI PLSAIFTT 37 PRT ArtificialP114AR7P92- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYLEFLL H5GGEAIVLTVPGSERSYDLTGLKPGTEYVVQIMGVKGGTVS PPLSAIFTT 38 PRT ArtificialP114AR7P93- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL D11GSGEAIVLTVPGSERSYDLTGLKPGTEYVVGINGVKGGYI SYPLSAIFTT 39 PRT ArtificialP114AR7P93- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL G8GSGEAIVLTVPGSERSYDLTDLKPGTEYGVTINGVKGGRV STPLSAIFTT 40 PRT ArtificialP114AR7P93- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL H9GSGEAIVLTVPGSERSYDLTGLKPGTEYVVQIIGVKGGHIS LPLSAIFTT 41 PRT ArtificialP114AR7P94- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL A3GSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKI SPPLSAIFTT 42 PRT ArtificialP114AR7P94- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL E5GSGEAIVLTVPGSERSYDLTGLKPGTEYAVNIMGVKGGRV SVPLSAIFTT 43 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL B9GSGEAIVLTVPGSERSYDLTGLKPGTEYVVQILGVKGGSI SVPLSAIFTT 44 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL D3GSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGSI SYPLSAIFTT 45 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL D4GSGEAIVLTVPGSERSYDLTGLKPGTEYVVQILGVKGGYI SIPLSAIFTT 46 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFL E3GSGEAIVLTVPGSERSYDLTGLKPGTEYVVQIMGVKGGTV SPPLSAIFTT 47 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFTT F10AGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGSIS PPLSAIFTT 48 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFELLS G7TGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGSIS PPLSAIFTT 49 PRT ArtificialP114AR7P95- LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFV H8SKGEAIVLIVPGSERSYDLTGLKPGTEYVVNIMGVKGGSI SPPLSAIFTT 50 PRT ArtificialECB1 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSIS VPLSAIFTT 51 PRT ArtificialECB2 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKIS PPLSAIFTT 52 PRT ArtificialECB3 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLIVPGSERSYDLTGLKPGTEYVVQIIGVKGGHIS LPLSAIFTT 53 PRT ArtificialECB4 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIRYDEFLRSGEAIVLTVPGSERSYDLTGLKPGTEYVVVTILGVKGGLVS TPLSAIFTT 54 PRT ArtificialECB5 MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKI SPPLSAIFTT 55 PRT ArtificialECB6 MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLIVPGSERSYDLTGLKPGTEYVVQIIGVKGGHIS LPLSAIFTT 56 PRT ArtificialECB7 MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLIVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGGSGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLIVPGSERSYDLTGLKPGTEYVVQIIGVKGGHIS LPLSAIFTT 57 PRT ArtificialECB15 MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKISPPLSAIFTT 58 PRT Artificial ECB27MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSISVPLSAIFTT 59 PRT Artificial ECB60MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKISPPLSAIFTT 60 PRT Artificial ECB37MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSISVPLSAIFTT 61 PRT Artificial ECB94MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKISPPLSAIFTT 62 PRT Artificial ECB95MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 63 PRT Artificial ECB96MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 64 PRT Artificial ECB97MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSVVTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 65 PRT Artificial ECB106MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKISPPLSAIFTT 66 PRT Artificial ECB107MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 67 PRT Artificial ECB108MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 68 PRT Artificial ECB109MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 69 PRT Artificial ECB118MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKISPPLSAIFTT 70 PRT Artificial ECB119MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 71 PRT Artificial ECB120MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 72 PRT Artificial ECB121MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT SEQ ID NO: 73, PRT, Homo Sapiens,EGFR 1 mrpsgtagaa llallaalcp asraleekkv cqgtsnkltq lgtfedhfls lqrmfnncev61 vlgnleityv qrnydlsflk tiqevagyvl ialntverip lenlqiirgn myyensyala 121vlsnydankt glkelpmrnl qeilhgavrf snnpalcnve siqwrdivss dflsnmsmdf 181qnhlgscqkc dpscpngscw gageencqkl tkiicaqqcs grcrgkspsd cchnqcaagc 241tgpresdclv crkfrdeatc kdtcpplmly npttyqmdvn pegkysfgat cvkkcprnyv 301vtdhgscvra cgadsyemee dgvrkckkce gperkvcngi gigefkdsls inatnikhfk 361nctsisgdlh ilpvafrgds fthtppldpq eldilktvke itgflliqaw penrtdlhaf 421enleiirgrt kqhgqfslav vslnitslgl rslkeisdgd viisgnknlc yantinwkkl 481fgtsgqktki isnrgensck atgqvchalc spegcwgpep rdcvscrnvs rgrecvdkcn 541llegeprefv enseciqchp eclpqamnit ctgrgpdnci qcahyidgph cvktcpagvm 601genntivwky adaghvchlc hpnctygctg pglegcptng pkipsiatgm vgalllllvv 661algiglfmrr rhivrkrtlr rllqerelve pltpsgeapn qallrilket efkkikvlgs 721gafgtvykgl wipegekvki pvaikelrea tspkankeil deayvmasvd nphvcrllgi 781cltstvqlit qlmpfgclld yvrehkdnig sqyllnwcvq iakgmnyled rrlvhrdlaa 841rnvlvktpqh vkitdfglak llgaeekeyh aeggkvpikw malesilhri ythqsdvwsy 901gvtvwelmtf gskpydgipa seissilekg erlpqppict idvymimvkc wmidadsrpk 961freliiefsk mardpqrylv iqgdermhlp sptdsnfyra lmdeedmddv vdadeylipq 1021qgffsspsts rtpllsslsa tsnnstvaci drnglqscpi kedsflqrys sdptgalted 1081siddtflpvp eyinqsvpkr pagsvqnpvy hnqpinpaps rdphyqdphs tavgnpeyln 1141tvqptcvnst fdspahwaqk gshqisldnp dyqqdffpke akpngifkgs taenaeylry 1201apqssefiga 74 PRT Homo EGF NSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGsapiens ERCQYRDLKWWELR SEQ ID NO: 75, PRT, Homo Sapiens, Tenascin-C 1mgamtqllag vflaflalat eggvlkkvir hkrqsgvnat lpeenqpvvf nhvyniklpv 61gsqcsvdles asgekdlapp sepsesfqeh tvdgenqivf thriniprra cgcaaapdvk 121ellsrleele nlvsslreqc tagagcclqp atgrldtrpf csgrgnfste gcgcvcepgw 181kgpncsepec pgnchlrgrc idgqcicddg ftgedcsqla cpsdcndqgk cvngvcicfe 241gyagadcsre icpvpcseeh gtcvdglcvc hdgfagddcn kplclnncyn rgrcvenecv 301cdegftgedc selicpndcf drgrcingtc yceegftged cgkptcphac htqgrceegq 361cvcdegfagv dcsekrcpad chnrgrcvdg rcecddgftg adcgelkcpn gcsghgrcvn 421gqcvcdegyt gedcsqlrcp ndchsrgrcv egkcvceqgf kgydcsdmsc pndchqhgrc 481vngmcvcddg ytgedcrdrq cprdcsnrgl cvdgqcvced gftgpdcael scpndchgqg 541rcvngqcvch egfmgkdcke qrcpsdchgq grcvdgqcic hegftgldcg qhscpsdcnn 601lgqcvsgrci cnegysgedc sevsppkdlv vtevteetvn lawdnemrvt eylvvytpth 661egglemqfry pgdqtstiiq elepgveyfi rvfailenkk sipvsarvat ylpapeglkf 721ksiketsvev ewdpldiafe tweiifrnmn kedegeitks lrrpetsyrq tglapgqeye 781islhivknnt rgpglkrvtt trldapsqie vkdvtdttal itwfkplaei dgieltygik 841dvpgdrttid ltedenqysi gnlkpdteye vslisrrgdm ssnpaketft tgldaprnlr 901rvsqtdnsit lewrngkaai dsyrikyapi sggdhaevdv pksqqattkt tltglrpgte 961ygigvsavke dkesnpatin aateldtpkd lqvsetaets ltllwktpla kfdryrinys 1021lptgqwvgvq lprnttsyvl rglepgqeyn vlltaekgrh kskparvkas teqapelenl 1081tvtevgwdgl rinwtaadqa yehfiiqvqe ankveaarnl tvpgslravd ipglkaatpy 1141tvsiygviqg yrtpvlsaea stgetpnlge vvvaevgwda lklnwtapeg ayeyffiqvq 1201eadtveaaqn ltvpgglrst dlpglkaath ytitirgvtq dfsttplsve vlteevpdmg 1261nitvtevswd alrinwttpd gtydqftiqv qeadqveeah nitvpgslrs meipglragt 1321pytvtlhgev rghstrplav evvtedlpql gdlaysevgw dglrinwtaa dnayehfviq 1381vqevnkveaa qnitlpgslr avdipgleaa tpyrvsiygv irgyrtpvls aeastakepe 1441ignlnvsdit pesfnlswma tdgifetfti eiidsnrlle tveynisgae rtahisglpp 1501stdfivylsg lapsirtkti satattealp llenitisdi npygftvswm asenafdsfl 1561vtvvdsgkll dpqeftlsgt qrklelrgli tgigyevmvs gftqghqtkp lraeivteae 1621pevdnllvsd atpdgfrlsw tadegvfdnf vlkirdtkkq sepleitlla pertrditgl 1681reateyeiel ygiskgrrsq tvsaiattam gspkevifsd itensatvsw raptaqvesf 1741rityvpitgg tpsmvtvdgt ktqtrlvkli pgveylvsii amkgfeesep vsgsfttald 1801gpsglvtani tdsealarwq paiatvdsyv isytgekvpe itrtvsgntv eyaltdlepa 1861teytlrifae kgpqksstit akfttdldsp rdltatevqs etalltwrpp rasvtgyllv 1921yesvdgtvke vivgpdttsy sladlspsth ytakicialn gplrsnmiqt ifttigllyp f 1981pkdcsqamln gdttsglyti ylngdkaeal evfcdmtsdg ggwivflrrk ngrenfyqnw 2041kayaagfgdr reefwlgldn lnkitaqgqy elrvdlrdhg etafavydkf svgdaktryk 2101lkvegysgta gdsmayhngr sfstfdkdtd saitncalsy kgafwyrnch rvnlmgrygd 2161nnhsqgvnwf hwkghehsiq faemklrpsn frnlegrrkr a 76 PRT Artificial FibconLdaptdlqvtnvtdtsitvswtppsatitgyritytpsngpgepkeltvppsstsvtitgltpgveyvvslyalkdnqespplvgtqtt 77 PRT Artificial 10th FN3 domain ofVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPV fibronectin (FN10)QEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINY RT 78 PRT ArtificialLinker GSGS 79 PRT Artificial Linker GGGGSGGGGSGGGGSGGGGSGGGGS 80 PRTArtificial Linker APAP 81 PRT Artificial Linker APAPAPAPAP 82 PRTArtificial Linker APAPAPAPAPAPAPAPAPAP 83 PRT Artificial LinkerAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPA PAP 84 PRT Artificial LinkerAEAAAKEAAAKEAAAKEAAAKEAAAKAAA 85 PRT Artificial Tencon BC loop TAPDAAFD86 PRT Artificial Tencon GF loop KGGHRSN 87 PRT Artificial P53A1R5-17 BCloop ADPHGFYD 88 PRT Artificial P54AR4-17 BC loop TYDRDGYD 89 PRTArtificial P54AR4-47 BC loop WDPFSFYD 90 PRT Artificial P54AR4-48 BCloop DDPRGFYE 91 PRT Artificial P54AR4-73 BC loop TWPYADLD 92 PRTArtificial P54AR4-74 BC loop GYNGDHFD 93 PRT Artificial P54AR4-81 BCloop DYDLGVYD 94 PRT Artificial P54AR4-83 BC loop DDPWDFYE 95 PRTArtificial FG loops of EGFR HNVYKDTNMRGL 96 PRT Artificial FG loops ofEGFR LGSYVFEHDVM 97 DNA Artificial >EGFR part ECB97;Atgttgccagcgccgaagaacctggtagttagcgaggttactgaggac P54AR4-83v22agcgcgcgtctgagctgggacgatccgtgggcgttctacgagagctttctgatccagtatcaagagagcgagaaagtcggtgaagcgattgtgctgaccgtcccgggctccgagcgttcctacgacctgaccggtttgaagccgggtaccgagtatacggtgagcatctacggtgttcacaatgtctataaggacactaatatccgcggtctgcctctgagcgccattttcaccacc 98 DNA Artificial >EGFR partECB15; Atgctgccagcccctaagaatctggtcgtgagcgaagtaaccgagga P54AR4-83v2cagcgcccgcctgagctgggacgacccgtgggcgttctatgagtctttcctgattcagtatcaagaaagcgaaaaagttggcgaagcgatcgtcctgaccgtcccgggtagcgagcgctcctacgatctgaccggcctgaaaccgggtacggagtacacggtgtccatttacggtglicacaatgtgtataaagacaccaacatgcgtggcctgccgctgtcggcgattttcaccacc 99 PRT Artificial tencon 27LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYG VKGGHRSNPLSAIFTT 100 PRTArtificial TCL14 library LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFXIXYXEXXXXGEAIVLTVPGSERSYDLTGLKPGTEYXVXIXG VKGGXXSXPLSAIFTT >SEQ ID NO: 101PRT Homo sapiens cMet 1 mkapavlapg ilvllftivq rsngeckeal aksemnvnmkyqlpnftaet piqnvilheh 61 hiflgatnyi yvineedlqk vaeyktgpvl ehpdcfpcqdcsskanlsgg vwkdninmal 121 vvdtyyddql iscgsvnrgt cqrhvfphnh tadiqsevhcifspqieeps qcpdcvvsal 181 gakvlssvkd rfinffvgnt inssyfpdhp lhsisvrrlketkdgfmflt dqsyidvlpe 241 frdsypikyv hafesnnfiy fltvqretld aqtfhtriirfcsinsglhs ymemplecil 301 tekrkkrstk kevfnilqaa yvskpgagla rqigaslnddilfgvfaqsk pdsaepmdrs 361 amcafpikyv ndffnkivnk nnvrclqhfy gpnhehcfnrtllrnssgce arrdeyrtef 421 ttalqrvdlf mgqfsevllt sistfikgdl tianlgtsegrfmqvvvsrs gpstphvnfl 481 ldshpvspev ivehtlnqng ytivitgkki tkipinglgcrhfqscsqcl sappfvqcgw 541 chdkcvrsee clsgtwtqqi clpaiykvfp nsapleggtrlticgwdfgf rrnnkfdlkk 601 trvllgnesc tltlsestmn tlkctvgpam nkhfnmsiiisnghgttqys tfsyvdpvit 661 sispkygpma ggtlltltgn ylnsgnsrhi siggktctlksysnsilecy tpaqtistef 721 avklkidlan retsifsyre dpivyeihpt ksfistwwkepinivsflfc fasggstitg 781 vgknlnsysv prmvinvhea grnftvacqh rsnseiiccttpslqqlnlq Lplktkaffm 841 ldgilskyfd liyvhnpvfk pfekpvmism gnenvleikgndidpeavkg evlkvgnksc 901 enihlhseav lctvpndllk lnselniewk qaisstvlgkvivqpdqnft gliagvvsis 961 talllllgff lwlkkrkqik dlgselvryd arvhtphldrlvsarsyspt temvsnesvd 1021 yratfpedqf pnssqngscr qvqypltdms piltsgdsdisspllqntvh idlsalnpel 1081 vqavqhvvig psslivhfne vigrghfgcv yhgtlldndgkkihcavksl nritdigevs 1141 qfltegiimk dfshpnvlsl lgiclrsegs plvvlpymkhgdlrnfirne thnptvkdli 1201 gfglqvakgm kylaskkfvh rdlaarncml dekftvkvadfglardmydk eyysvhnktg 1261 aklpvkwmal eslqtqkftt ksdvwsfgvl lwelmtrgappypdvntfdi tvyllqgrrl 1321 lqpeycpdpl yevmlkcwhp kaemrpsfse lvsrisaifstfigehyvhv natyvnvkcv 1381 apypsllsse dnaddevdtr pasfwets 102 PRT HomoHGF QRKRRNTIHEFKKSAKTTLIKIDPALKIK sapiensTKKVNTADQCANRCTRNKGLPFTCKAFVFDKARKQCLWFPFNSMS SGVKKEFGHEFDLYENKDYRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEHSFLPSSYRG KDLQENYCRNPRGEEGGPVVCFTSNPEVRYEVCDIPQCSEVECMTCNGESYRGLMDH TESGKICQRVVDHQTPHRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRWEYCAIK TCADNTMNDTDVPLETTECIQGQGEGYRGTVNTIVVNGIPCQRVVDSQYPHEHDMTPENFKC KDLRENYCRNPDGSESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYMGNLSQT RSGLTCSMVVDKNMEDLHRHIFVVEPDASKLNENYCRNPDDDAHGPVVCYTGNPLIPVVDYCPIS RCEGDTTPTIVNLDHPVISCAKTKQLRVVNGIPTRTNIGVVMVSLRYRNKHICGGSLIKESW VLTARQCFPSRDLKDYEAVVLGIHDVHGRGDEKCKQVLNVSQLVYGPEGSDLVLMKLAR PAVLDDFVSTIDLPNYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNEKCSQHHRG KVTLNESEICAGAEKIGSGPCEGDYGGPLVCEQHKMRMVLGVIVPGRGCAIPNRPGIFV RVAYYAKWIHKII LTYKVPQS103 DNA Artificial >cMET part ECB97Ctgccggctccgaagaacttggtggtgagccgtgttaccgaagatagc P114AR7P95-C5v2gcacgcctgagctggacggcaccggatgcggcgttcgatagcttctggattcgctattttgagtttctgggtagcggtgaggcaattgttctgacggtgccgggctctgaacgctcctacgatttgaccggtctgaaaccgggcaccgagtatgtggtgaacattctgagcgttaagggcggtagcatcagcccaccgctgagcgcgatcttcacgactggtggttgc 104 DNA Artificial >cMET part ECB15Ctgccggcaccgaagaacctggttgtcagccgtgtgaccgaggatag P114AR7P94-A3cgcacgtttgagctggaccgctccggatgcagcctttgacagcttctggattcgttactttgaatttctgggtagcggtgaggcgatcgttctgacggtgccgggctctgaacgcagctatgatttgacgggcctgaagccgggtactgagtacgtggttaacatcatgggcgttaagggtggtaaaatcagcccgccatt gtccgcgatctttaccacg105 PRT Artificial linker GGGGS 106 PRT Artificial ECB91mlpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnirglplsaifttapapapapapLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 107 PRT Artificial P53A1R5-17v2lpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnmrglplsaiftt 108 PRT Artificial P54AR4-83v22lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnirglplsaiftt 109 PRT Artificial P54AR4-83v23lpapknlvvsevtedsarlswddphafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnirglplsaiftt 110 PRT Artificial P53A1R5-17v22lpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnirglplsaiftt 111 PRT ArtificialP114AR7P94-A3v22lpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnilgvkggkispplsaiftt 112 PRT Artificial P114AR9P121-A6v2LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 113 PRT ArtificialP114AR9P122-A7v2 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLIVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 114 PRT ArtificialP114AR7P95-05v2 LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 115 DNA Artificial ECB97atgttgccagcgccgaagaacctggtagttagcgaggttactgaggacagcgcgcgtctgagctgggacgatccgtgggcgttctacgagagctttctgatccagtatcaagagagcgagaaagtcggtgaagcgattgtgctgaccgtcccgggctccgagcgttcctacgacctgaccggtttgaagccgggtaccgagtatacggtgagcatctacggtgttcacaatgtctataaggacactaatatccgcggtctgcctctgagcgccattttcaccaccgcaccggcaccggctccggctcctgccccgctgccggctccgaagaacttggtggtgagccgtgttaccgaagatagcgcacgcctgagctggacggcaccggatgcggcgttcgatagcttctggattcgctattttgagtttctgggtagcggtgaggcaattgttctgacggtgccgggctctgaacgctcctacgatttgaccggtctgaaaccgggcaccgagtatgtggtgaacattctgagcgttaagggcggtagcatcagcccaccgctgagcgcgatcttcacgactggtggttgc 116 DNA ArtificialECB15 atgctgccagcccctaagaatctggtcgtgagcgaagtaaccgaggacagcgcccgcctgagctgggacgacccgtgggcgttctatgagtctttcctgattcagtatcaagaaagcgaaaaagttggcgaagcgatcgtcctgaccgtcccgggtagcgagcgctcctacgatctgaccggcctgaaaccgggtacggagtacacggtgtccatttacggtgttcacaatgtgtataaagacaccaacatgcgtggcctgccgctgtcggcgattttcaccaccgcgcctgcgccagcgcctgcaccggctccgctgccggcaccgaagaacctggttgtcagccgtgtgaccgaggatagcgcacgtttgagctggaccgctccggatgcagcctttgacagcttctggattcgttactttgaatttctgggtagcggtgaggcgatcgttctgacggtgccgggctctgaacgcagctatgatttgacgggcctgaagccgggtactgagtacgtggttaacatcatgggcgttaagggtggtaaaatcagcccgccattgtccgcgatctttaccacg 117 PRT Artificial albuminbinding tidewIlkeakekaieelkkagitsdyyfdlinkaktvegvnalkdeilka domain 118PRT Artificial ECB18 nnlpapknlvvsevtedsarlswddpwafyesfligygesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwirydevvvggeaivltvpgsersydltglkpgteyyvnilgvkggsisvplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 119 PRT Artificial ECB28nnlpapknlvvsevtedsarlswadphgfydsfligygesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwirydevvvggeaivltvpgsersydltglkpgteyyvnilgvkggsisvplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 120 PRT Artificial ECB38nnlpapknlvvsevtedsarlswddpwafyesfligygesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkggkispplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 121 PRT Artificial ECB39nnlpapknlvvsevtedsarlswadphgfydsfligygesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkggkispplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 122 PRT ArtificialP53A1R5-17 wthMet MLPAPKNLVVSEVTEDSLRLSWADPHGFYDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 123 PRTArtificial P54AR4-17 with Met MLPAPKNLVVSEVTEDSLRLSWTYDRDGYDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 124 PRTArtificial P54AR4-47 with Met MLPAPKNLVVSEVTEDSLRLSWGYNGDHFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 125 PRTArtificial P54AR4-48 with Met MLPAPKNLVVSEVTEDSLRLSWDDPRGFYESFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 126 PRTArtificial P54AR4-73 with Met MLPAPKNLVVSEVTEDSLRLSVVTVVPYADLDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 127 PRTArtificial 54AR4-74 with Met MLPAPKNLVVSEVTEDSLRLSWGYNGDHFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 128 PRTArtificial P54AR4-81 with Met MLPAPKNLVVSEVTEDSLRLSWDYDLGVYFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSI YGVHNVYKDTNMRGLPLSAEFTT 129 PRTArtificial P54AR4-83 with Met MLPAPKNLVVSEVTEDSLRLSWDDPWAFYESFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 130 PRTArtificial P54CR4-31 with Met MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVLGSYVFEHDVMLPLSAEFTT 131 PRTArtificial P54AR4-83v2 with MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQY MetQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAIFIT 132 PRTArtificial P54CR4-31v2 with MLPAPKNLVVSEVTEDSARLSVVTAPDAAFDSFLIQY MetQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIY GVLGSYVFEHDVMLPLSAIFTT 133 PRTArtificial P54AR4-73v2 MLPAPKNLVVSEVTEDSLRLSWTWPYADLDSFLIQY withMetQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIY GVHNVYKDTNMRGLPLSAEFTT 134 PRTArtificial P53A1R5-17v2 withmIpapknIvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgser MetsydItgIkpgteytvsiygvhnvykdtnmrglplsaiftt 135 PRT Artificial P54AR4-83v22with mIpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgse Metrsydltglkpgteytvsiygvhnvykdtnirglplsaiftt 136 PRT ArtificialP54AR4-83v23 withmIpapknlvvsevtedsarlswddphafyesfliqyqesekvgeaivltvpgser MetsydItgIkpgteytvsiygvhnvykdtnirgIplsaiftt 137 PRT ArtificialP53A1R5-17v22 withmIpapknIvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgser MetsydItgIkpgteytvsiygvhnvykdtnirgIplsaiftt 138 PRT Artificial ECB1 withoutMet LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPG TEYYVNILGVKGGSISVPLSAIFTT 139 PRTArtificial ECB2 without Met LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGT EYWNIMGVKGGKISPPLSAIFTT 140 PRTArtificial ECB3 without Met LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPG TEYWQIIGVKGGHISLPLSAIFTT 141 PRTArtificial ECB4 without Met LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIRYDEFLRSGEAIVLTVPGSERSYDLTGLKPGT EYWVTILGVKGGLVSTPLSAIFTT 142 PRTArtificial ECB5 without Met LPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPG TEYWNIMGVKGGKISPPLSAIFTT 143 PRTArtificial ECB6 without Met LPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPG TEYWQIIGVKGGHISLPLSAIFTT 144 PRTArtificial ECB7 without Met LPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTGGGGSGGGGSGGGG SGGGGSMLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPG TEYWQIIGVKGGHISLPLSAIFTT 145 PRTArtificial ECB15 without Met LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKI SPPLSAIFTT 146 PRT ArtificialECB27 without Met LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSI SVPLSAIFTT 147 PRT ArtificialECB60 without Met LPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPMLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGS GEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKISPPLSAIFTT 148 PRT Artificial ECB37 without MetLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYDEVVVGGEAIVLTVPGSERSYDLTGLKPGTEYYVNILGVKGGSI SVPLSAIFTT 149 PRT ArtificialECB94 without Met LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKIS PPLSAIFTT 150 PRT Artificial ECB95without Met LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSG EAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 151 PRT Artificial ECB96 without MetLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSIS PPLSAIFTT 152 PRT Artificial ECB97without Met LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISP PLSAIFTT 153 PRT ArtificialECB106 without Met LPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKIS PPLSAIFTT 154 PRT ArtificialECB107 without Met LPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSG EAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSISPPLSAIFTT 155 PRT Artificial ECB108 without MetLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSIS PPLSAIFTT 156 PRT ArtificialECB109 without Met LPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISP PLSAIFTT 157 PRT ArtificialECB118 without Met LPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKIS PPLSAIFTT 158 PRT ArtificialECB119 without Met LPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSG EAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSISPPLSAIFTT 159 PRT Artificial ECB120 without MetLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSIS PPLSAIFTT 160 PRT ArtificialECB121 without Met LPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISP PLSAIFTT 161 PRT Artificial ECB91without Met lpapknlvvsevtedsarlswddpwafyesfliqyqesekygeaiyltvpgsersydltglkpgteytvsiygvhnvykdtnirglplsaifttapapapapapLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTT 162 PRT Artificial ECB18 withoutMet lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwirydevvvggeaivltvpgsersydltglkpgteyyvnilgvkggsisvplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 163 PRT Artificial ECB28without Met lpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnmrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwirydevvvggeaivltvpgsersydltglkpgteyyvnilgvkggsisvplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 164 PRT Artificial ECB38without Met lpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkggkispplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 165 PRT Artificial ECB39without Met lpapknlvvsevtedsarlswadphgfydsfliqyqesekvgeaivltvpgsersydltglkpgteytvsiygvhnvykdtnnnrglplsaifttapapapapaplpapknlvvsrvtedsarlswtapdaafdsfwiryfeflgsgeaivltvpgsersydltglkpgteyvvnimgvkggkispplsaifttapapapapaplaeakvlanreldkygvsdyyknlinnaktvegvkalldeilaalp 166 DNA Artificial ECB97without Met ttgccagcgccgaagaacctggtagttagcgaggttactgaggacagcgcgcgtctgagctgggacgatccgtgggcgttctacgagagctttctgatccagtatcaagagagcgagaaagtcggtgaagcgattgtgctgaccgtcccgggctccgagcgttcctacgacctgaccggtttgaagccgggtaccgagtatacggtgagcatctacggtgttcacaatgtctataaggacactaatatccgcggtctgcctctgagcgccattttcaccaccgcaccggcaccggctccggctcctgccccgctgccggctccgaagaacttggtggtgagccgtgttaccgaagatagcgcacgcctgagctggacggcaccggatgcggcgttcgatagcttctggattcgctattttgagtttctgggtagcggtgaggcaattgttctgacggtgccgggctctgaacgctcctacgatttgaccggtctgaaaccgggcaccgagtatgtggtgaacattctgagcgttaagggcggtagcatcagcccaccgctgagcgcgatcttcacgactggtggttgc 167 DNA Artificial ECB15without Met ctgccagcccctaagaatctggtcgtgagcgaagtaaccgaggacagcgcccgcctgagctgggacgacccgtgggcgttctatgagtctttcctgattcagtatcaagaaagcgaaaaagttggcgaagcgatcgtcctgaccgtcccgggtagcgagcgctcctacgatctgaccggcctgaaaccgggtacggagtacacggtgtccatttacggtgttcacaatgtgtataaagacaccaacatgcgtggcctgccgctgtcggcgattttcaccaccgcgcctgcgccagcgcctgcaccggctccgctgccggcaccgaagaacctggttgtcagccgtgtgaccgaggatagcgcacgtttgagctggaccgctccggatgcagcctttgacagcttctggattcgttactttgaatttctgggtagcggtgaggcgatcgttctgacggtgccgggctctgaacgcagctatgatttgacgggcctgaagccgggtactgagtacgtggttaacatcatgggcgttaagggtggtaaaatcagcccgccattgtccgcgatctttaccacg 168 DNA Artificial >EGFR partECB97; ttgccagcgccgaagaacctggtagttagcgaggttactgaggacagc P54AR4-83v22gcgcgtctgagctgggacgatccgtgggcgttctacgagagctttctgat without metccagtatcaagagagcgagaaagtcggtgaagcgattgtgctgaccgtcccgggctccgagcgttcctacgacctgaccggtttgaagccgggtaccgagtatacggtgagcatctacggtgttcacaatgtctataaggacactaatatccgcggtctgcctctgagcgccattttcaccacc 169 DNA Artificial >EGFR partECB15; ctgccagcccctaagaatctggtcgtgagcgaagtaaccgaggacag P54AR4-83v2cgcccgcctgagctgggacgacccgtgggcgttctatgagtctttcctga without Metttcagtatcaagaaagcgaaaaagttggcgaagcgatcgtcctgaccgtcccgggtagcgagcgctcctacgatctgaccggcctgaaaccgggtacggagtacacggtgtccatttacggtgttcacaatgtgtataaagacaccaacatgcgtggcctgccgctgtcggcgattttcaccacc 170 PRT Artificial ECB94 withC-ter MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSG EAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGKISPPLSAIFTTC 171 PRT Artificial ECB95 with C-terMLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLWSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSI SPPLSAIFTTC 172 PRT ArtificialECB96 with C-ter MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSI SPPLSAIFTTC 173 PRT ArtificialECB97 with C-ter MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYWNILSVKGGSIS PPLSAIFTTC 174 PRT ArtificialECB106 with C-ter MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGKI SPPLSAIFTTC 175 PRT ArtificialECB107 with C-ter MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVGSGEAIVLTVPGSERSYDLTGLKPGTEYWNILGVKGGSI SPPLSAIFTTC 176 PRT ArtificialECB108 with C-ter MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFVSKGDAIVLTVPGSERSYDLTGLKPGTEYVVNILGVKGGSI SPPLSAIFTTC 177 PRT ArtificialECB109 with C-ter MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQY cysteineQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYWNILSVKGGSIS PPLSAIFTTC 178 PRT ArtificialECB91 with C-ter mlpapknlvvsevtedsarlswddpwafyesfliqyqesekvgeaivltvpgsecysteine rsydltglkpgteytvsiygvhnvykdtnirglplsaifttapapapapapLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNILSVKGGSISPPLSAIFTTC >SEQ ID NO: 179 PRT ArtificialAn FG loop of EGFR binding FN3 domain HNVYKDTNX₉RGL; wherein X₉ is M orI >SEQ ID NO: 180 PRT Artificial A FG loop of EGFR binding FN3 domainLGSYVFEHDVML (SEQ ID NO: 180), >SEQ ID NO: 181 PRT Artificial a BC loopof EGFR binding FN3 domain X₁X₂X₃X₄X₅X₆X₇X₈ (SEQ ID NO: 181), wherein X₁is A, T, G or D; X₂ is A, D, Y or W; X₃ is P, D or N; X₄ is L or absent;X₅ is D, H, R, G, Y or W; X₆ is G, D or A; X₇ is A, F, G, H or D; and X₈is Y, F or L. >SEQ ID NO: 182 PRT Artificial EGFR binding FN3 domainLPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNX₉RGLPLSAEFTT (SEQ ID NO: 182), X₁ is A,T, G or D; X₂ is A, D, Y or W; X₃ is P, D or N; X₄ is L or absent; X₅ isD, H, R, G, Y or W; X₆ is G, D or A; X₇ is A, F, G, H or D; X₈ is Y, For L; and X₉ is M or I >SEQ ID NO: 183 PRT Artificial EGFR binding FN3domain LPAPKNLVVSEVTEDSLRLSWX₁X₂X₃X₄X₅X₆X₇X₈DSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSYVFEHDVMLPLSAEFTT (SEQ ID NO: 183), whereinX₁ is A, T, G or D; X₂ is A, D, Y or W; X₃ is P, D or N; X₄ is L orabsent; X₅ is D, H, R, G, Y or W; X₆ is G, D or A; X₇ is A, F, G, H orD; and X₈ is Y, F or L. >SEQ ID NO: 184 PRT Artificial A C-met bindingFN3 domain C strand and a CD loop sequence DSFX₁₀IRYX₁₁EX₁₂X₁₃X₁₄X₁₅GX₁₆ (SEQ ID NO: 184), wherein X₁₀ is W, F or V; X₁₁ is D, For L; X₁₂ is V, F or L; X₁₃ is V, L or T; X₁₄ is V, R, G, L, T or S; X₁₅is G, S, A, T or K; and X₁₆ is E or D; and >SEQ ID NO: 185 PRTArtificial A c-Met binding FN3 domain F strand and a FG loopTEYX₁₇VX₁₈IX₁₉X₂₀V KGGX₂₁X₂₂SX₂₃ (SEQ ID NO: 185), wherein X₁₇ is Y, W,I, V, G or A; X₁₈ is N, T, Q or G; X₁₉ is L, M, N or I; X₂₀ is G or S;X₂₁ is S, L, G, Y, T, R, H or K; X₂₂ is I, V or L; and X₂₃ is V, T, H,I, P, Y, T or L. >SEQ ID NO: 186 PRT Artificial a c-Met binding FN3domain LPAPKNLVVSRVTEDSARLSWTAPDAAF DSFX₁₀IRYX₁₁E X₁₂X₁₃X₁₄X₁₅GX₁₆AIVLTVPGSERSYDLTGLKPGTEYX₁₇VX₁₈IX₁₉X₂₀VKGGX₂₁X₂₂SX₂₃PLSAEFTT (SEQ ID NO:186), wherein X₁₀ is W, F or V; and X₁₁ is D, F or L; X₁₂ is V, F or L;X₁₃ is V, L or T; X₁₄ is V, R, G, L, T or S; X₁₅ is G, S, A, T or K; X₁₆is E or D; X₁₇ is Y, W, I, V, G or A; X₁₈ is N, T, Q or G; X₁₉ is L, M,N or I; X₂₀ is G or S; X₂₁ is S, L, G, Y, T, R, H or K; X₂₂ is I, V orL; and X₂₃ is V, T, H, I, P, Y, T or L. >SEQ ID NO: 187 PRT ArtificialEGFR FN3 domain of a bispecific EGFR/c-Met FN3 domain containingmolecule LPAPKNLVVSX₂₄VTX₂₅DSX₂₆RLSWDDPX₂₇AFYX₂₈SFLIQYQX₂₉SEKVGEAIX₃₀LTVPGSERSYDLTGLKPGTEYTVSIYX₃₁VHNVYKDTNX₃₂RGLPLSAX₃₃FTT (SEQ ID NO: 187),wherein X₂₄ is E, N or R; X₂₅ is E or P; X₂₆ is L or A; X₂₇ is H or W;X₂₈ is E or D; X₂₉ is E or P; X₃₀ is N or V; X₃₁ is G or Y; X₃₂ is M orI; and X₃₃ is E or I; >SEQ ID NO: 188 c-Met FN3 domain of a bispecificEGFR/c-Met FN3 domain containing moleculeLPAPKNLVVSX₃₄VTX₃₅DSX₃₆RLSWTAPDAAFDSFWIRYFX₃₇FX₃₈X₃₉X₄₀GX₄₁AIX₄₂LTVPGSERSYDLTGLKPGTEYVVNIX₄₃X₄₄VKGGX₄₅ISPPLSAX₄₆FTT (SEQ ID NO: 188);wherein X₃₄ is E, N or R; X₃₅ is E or P; X₃₆ is L or A; X₃₇ is E or P;X₃₈ is V or L; X₃₉ is G or S; X₄₀ is S or K; X₄₁ is E or D; X₄₂ is N orV; X₄₃ is L or M; X₄₄ is G or S; X₄₅ is S or K; and X₄₆ is E or I. 189PRT Artificial P54AR4- MLPAPKNLCVSEVTEDSARLSWDDPWAF 83v2-V9CYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTT 190 PRT Artificial P54AR4-MLPAPKNLVVCEVTEDSARLSWDDPWAF 83v2-S11C YESFLIQYQESEKVGEAIVLTVPGSERSYDLwith TGLKPGTEYTVSIYGVHNVYKDTNMRGLP methionine LSAIFTT 191 PRT ArtificialP54AR4- MLPAPKNLVVSCVTEDSARLSWDDPWAF 83v2-E12CYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTT 192 PRT Artificial P54AR4-MLPAPKNLVVSEVTCDSARLSWDDPWAF 83v2-E15C YESFLIQYQESEKVGEAIVLTVPGSERSYDLwith TGLKPGTEYTVSIYGVHNVYKDTNMRGLP methionine LSAIFTT 193 PRT ArtificialP54AR4- MLPAPKNLVVSEVTECSARLSWDDPWAF 83v2-D16CYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTT 194 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDCARLSWDDPWAF 83v2-S17C YESFLIQYQESEKVGEAIVLTVPGSERSYDLwith TGLKPGTEYTVSIYGVHNVYKDTNMRGLP methionine LSAIFTT 195 PRT ArtificialP54AR4- MLPAPKNLVVSEVTEDSARLCWDDPWAF 83v2-S21CYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTT 196 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-S31C YECFLIQYQESEKVGEAIVLTVPGSERSYDwith LTGLKPGTEYTVSIYGVHNVYKDTNMRGL methionine PLSAIFTT 197 PRTArtificial P54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-Q35CYESFLICYQESEKVGEAIVLTVPGSERSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTT 198 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-S39C YESFLIQYQECEKVGEAIVLTVPGSERSYDwith LTGLKPGTEYTVSIYGVHNVYKDTNMRGL methionine PLSAIFTT 199 PRTArtificial P54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-K41CYESFLIQYQESECVGEAIVLTVPGSERSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTT 200 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-V42C YESFLIQYQESEKCGEAIVLTVPGSERSYDLwith TGLKPGTEYTVSIYGVHNVYKDTNMRGLP methionine LSAIFTT 201 PRT ArtificialP54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-I46CYESFLIQYQESEKVGEACVLTVPGSERSYD with LTGLKPGTEYTVSIYGVHNVYKDTNMRGLmethionine PLSAIFTT 202 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-L48C YESFLIQYQESEKVGEAIVCTVPGSERSYDwith LTGLKPGTEYTVSIYGVHNVYKDTNMRGL methionine PLSAIFTT 203 PRTArtificial P54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-T49CYESFLIQYQESEKVGEAIVLCVPGSERSYD with LTGLKPGTEYTVSIYGVHNVYKDTNMRGLmethionine PLSAIFTT 204 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-E54C YESFLIQYQESEKVGEAIVLTVPGSCRSYDwith LTGLKPGTEYTVSIYGVHNVYKDTNMRGL methionine PLSAIFTT 205 PRTArtificial P54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-R55CYESFLIQYQESEKVGEAIVLTVPGSECSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTT 206 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-T60C YESFLIQYQESEKVGEAIVLTVPGSERSYDLwith CGLKPGTEYTVSIYGVHNVYKDTNMRGLP methionine LSAIFTT 207 PRT ArtificialP54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-G61CYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TCLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTT 208 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-K63C YESFLIQYQESEKVGEAIVLTVPGSERSYDLwith TGLCPGTEYTVSIYGVHNVYKDTNMRGLP methionine LSAIFTT 209 PRT ArtificialP54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-G65CYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TGLKPCTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTT 210 PRT Artificial P54AR4-MLPAPKCLVVSEVTEDSARLSWDDPWAF 83v2-N7C YESFLIQYQESEKVGEAIVLTVPGSERSYDLwith TGLKPGTEYTVSIYGVHNVYKDTNMRGLP methionine LSAIFTT 211 PRT ArtificialP54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-S71CYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TGLKPGTEYTVCIYGVHNVYKDTNMRGLPmethionine LSAIFTT 212 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-L89C YESFLIQYQESEKVGEAIVLTVPGSERSYDLwith TGLKPGTEYTVSIYGVHNVYKDTNMRGLP methionine CSAIFTT 213 PRT ArtificialP54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-S90CYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LCAIFTT 214 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2A91C YESFLIQYQESEKVGEAIVLTVPGSERSYDLwith TGLKPGTEYTVSIYGVHNVYKDTNMRGLP methionine LSCIFTT 215 PRT ArtificialP54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-I92CYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSACFTT 216 PRT Artificial P54AR4-MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-T94C YESFLIQYQESEKVGEAIVLTVPGSERSYDLwith TGLKPGTEYTVSIYGVHNVYKDTNMRGLP methionine LSAIFCT 217 PRT ArtificialP54AR4- MLPAPKNLVVSEVTEDSARLSWDDPWAF 83v2-cysYESFLIQYQESEKVGEAIVLTVPGSERSYDL with TGLKPGTEYTVSIYGVHNVYKDTNMRGLPmethionine LSAIFTTGGHHHHHHC 218 PRT Artificial ECB147 withMLPAPKNLVVSEVTEDSARLSWDDPWAF methionine YESFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP LSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA IVLTVPGSERSYDLTGLKPGTEYVVNIMSVKGGSISPPLSAIFTTAPSPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 219 PRT Artificial ECB147v1 MLPAPKNLVVSEVTEDSARLSWDDPWAFwith YESFLIQYQESEKVGEAIVLTVPGSERSYDL methionineTGLKPGTEYTVSIYGVHNVYKDTNMRGLP LSAIFTTAPCPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA IVLTVPGSERSYDLTGLKPGTEYVVNIMSVKGGSISPPLSAIFTTAPSPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 220 PRT Artificial ECB147v2 MLPAPKNLVVSEVTEDSARLSWDDPWAFwith YESFLIQYQESEKVGEAIVLTVPGSERSYDL methionineTGLKPGTEYTVSIYGVHNVYKDTNMRGLP LSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA IVLTVPGSERSYDLTGLCPGTEYVVNIMSVKGGSISPPLSAIFTTAPAPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 221 PRT Artificial ECB147v3 MLPAPKNLVVSEVTEDSARLSWDDPWAFwith YESFLIQYQESEKVGEAIVLTVPGSERSYDL methionineTGLCPGTEYTVSIYGVHNVYKDTNMRGLP LSAIFTTAPCPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA IVLTVPGSERSYDLTGLCPGTEYVVNIMSVKGGSISPPLSAIFTTAPCPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 222 PRT Artificial ECB147v4 MLPAPKNLVVSEVTEDSARLSWDDPWAFwith YESFLIQYQESEKVGEAIVLTVPGSERSYDL methionineTGLKPGTEYTVSIYGVHNVYKDTNMRGLP LSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA IVLTVPGSERSYDLTGLKPGTEYVVNIMSVKGGSISPPLSAIFTTAPCPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 223 PRT Artificial ECB147v5 MLPAPKNLVVSEVTEDSARLSWDDPWAFwith YESFLIQYQESEKVGEAIVLTVPGSERSYDL methionineTGLCPGTEYTVSIYGVHNVYKDTNMRGLP LSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA IVLTVPGSERSYDLTGLCPGTEYVVNIMSVKGGSISPPLSAIFTTAPAPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 224 PRT Artificial ECB147v6 MLPAPKNLVVSEVTEDSARLSWDDPWAFwith YESFLIQYQESEKVGEAIVLTVPGSERSYDL methionineTGLCPGTEYTVSIYGVHNVYKDTNMRGLP LSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA IVLTVPGSERSYDLTGLKPGTEYVVNIMSVKGGSISPPLSAIFTTAPAPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 225 PRT Artificial ECB147v7 MLPAPKNLVVSEVTEDSARLSWDDPWAFwith YESFLIQYQESEKVGEAIVLTVPGSERSYDL methionineTGLKPGTEYTVSIYGVHNVYKDTNMRGLP LSAIFTTAPCPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA IVLTVPGSERSYDLTGLKPGTEYVVNIMSVKGGSISPPLSAIFTTAPCPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 226 PRT Artificial ECB82-cys MLPAPKNLVVSEVTEDSARLSWDDPWAFwith YESFLIQYQESEKVGEAIVLTVPGSERSYDL methionineTGLKPGTEYTVSIYGVHNVYKDTNMRGLP LSAIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEA IVLTVPGSERSYDLTGLKPGTEYVVNIMGVKGGKISPPLSAIFTTAPAPAPAPAPTIDEWL LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKAGGHHHHHHC 227 PRT Artificial P54AR4-LPAPKNLCVSEVTEDSARLSWDDPWAFYE 83v2-V8C SFLIQYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTT 228 PRTArtificial P54AR4- LPAPKNLVVCEVTEDSARLSWDDPWAFYE 83v2-S10CSFLIQYQESEKVGEAIVLTVPGSERSYDLTG without LKPGTEYTVSIYGVHNVYKDTNMRGLPLSmethionine AIFTT 229 PRT Artificial P54AR4-LPAPKNLVVSCVTEDSARLSWDDPWAFYE 83v2-E11C SFLIQYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTT 230 PRTArtificial P54AR4- LPAPKNLVVSEVTCDSARLSWDDPWAFYE 83v2-E14CSFLIQYQESEKVGEAIVLTVPGSERSYDLTG without LKPGTEYTVSIYGVHNVYKDTNMRGLPLSmethionine AIFTT 231 PRT Artificial P54AR4-LPAPKNLVVSEVTECSARLSWDDPWAFYE 83v2-D15C SFLIQYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTT 232 PRTArtificial P54AR4- LPAPKNLVVSEVTEDCARLSWDDPWAFYE 83v2-S16CSFLIQYQESEKVGEAIVLTVPGSERSYDLTG without LKPGTEYTVSIYGVHNVYKDTNMRGLPLSmethionine AIFTT 233 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLCWDDPWAFYE 83v2-S20C SFLIQYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTT 234 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-S30CCFLIQYQESEKVGEAIVLTVPGSERSYDLT without GLKPGTEYTVSIYGVHNVYKDTNMRGLPLmethionine SAIFTT 235 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-Q34C SFLICYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTT 236 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-S38CSFLIQYQECEKVGEAIVLTVPGSERSYDLT without GLKPGTEYTVSIYGVHNVYKDTNMRGLPLmethionine SAIFTT 237 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-K40C SFLIQYQESECVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTT 238 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-V41CSFLIQYQESEKCGEAIVLTVPGSERSYDLTG without LKPGTEYTVSIYGVHNVYKDTNMRGLPLSmethionine AIFTT 239 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-I45C SFLIQYQESEKVGEACVLTVPGSERSYDLTwithout GLKPGTEYTVSIYGVHNVYKDTNMRGLPL methionine SAIFTT 240 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-L47CSFLIQYQESEKVGEAIVCTVPGSERSYDLT without GLKPGTEYTVSIYGVHNVYKDTNMRGLPLmethionine SAIFTT 241 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-T48C SFLIQYQESEKVGEAIVLCVPGSERSYDLTwithout GLKPGTEYTVSIYGVHNVYKDTNMRGLPL methionine SAIFTT 242 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-E53CSFLIQYQESEKVGEAIVLTVPGSCRSYDLT without GLKPGTEYTVSIYGVHNVYKDTNMRGLPLmethionine SAIFTT 243 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-R54C SFLIQYQESEKVGEAIVLTVPGSECSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTT 244 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-T59CSFLIQYQESEKVGEAIVLTVPGSERSYDLC without GLKPGTEYTVSIYGVHNVYKDTNMRGLPLmethionine SAIFTT 245 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-G60C SFLIQYQESEKVGEAIVLTVPGSERSYDLTCwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTT 246 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-K62CSFLIQYQESEKVGEAIVLTVPGSERSYDLTG without LCPGTEYTVSIYGVHNVYKDTNMRGLPLSmethionine AIFTT 247 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-G64C SFLIQYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPCTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTT 248 PRTArtificial P54AR4- LPAPKCLVVSEVTEDSARLSWDDPWAFYE 83v2-N6CSFLIQYQESEKVGEAIVLTVPGSERSYDLTG without LKPGTEYTVSIYGVHNVYKDTNMRGLPLSmethionine AIFTT 249 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-S70C SFLIQYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVCIYGVHNVYKDTNMRGLPLS methionine AIFTT 250 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-L88CSFLIQYQESEKVGEAIVLTVPGSERSYDLTG without LKPGTEYTVSIYGVHNVYKDTNMRGLPCSmethionine AIFTT 251 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-S89C SFLIQYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLC methionine AIFTT 252 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2A90CSFLIQYQESEKVGEAIVLTVPGSERSYDLTG without LKPGTEYTVSIYGVHNVYKDTNMRGLPLSmethionine CIFTT 253 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-I91C SFLIQYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine ACFTT 254 PRTArtificial P54AR4- LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-T93CSFLIQYQESEKVGEAIVLTVPGSERSYDLTG without LKPGTEYTVSIYGVHNVYKDTNMRGLPLSmethionine AIFCT 255 PRT Artificial P54AR4-LPAPKNLVVSEVTEDSARLSWDDPWAFYE 83v2-cys SFLIQYQESEKVGEAIVLTVPGSERSYDLTGwithout LKPGTEYTVSIYGVHNVYKDTNMRGLPLS methionine AIFTTGGHHHHHHC 256 PRTArtificial ECB147 LPAPKNLVVSEVTEDSARLSWDDPWAFYE withoutSFLIQYQESEKVGEAIVLTVPGSERSYDLTG methionine LKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTED SARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMSV KGGSISPPLSAIFTTAPSPAPAPAPLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGV KALLDEILAALP 257 PRT Artificial ECB147v1LPAPKNLVVSEVTEDSARLSWDDPWAFYE without SFLIQYQESEKVGEAIVLTVPGSERSYDLTGmethionine LKPGTEYTVSIYGVHNVYKDTNMRGLPLS AIFTTAPCPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIV LTVPGSERSYDLTGLKPGTEYVVNIMSVKGGSISPPLSAIFTTAPSPAPAPAPLAEAKVL ANRELDKYGVSDYYKNLINNAKTVEGVK ALLDEILAALP258 PRT Artificial ECB147v2 LPAPKNLVVSEVTEDSARLSWDDPWAFYE withoutSFLIQYQESEKVGEAIVLTVPGSERSYDLTG methionine LKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTED SARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLCPGTEYVVNIMSV KGGSISPPLSAIFTTAPAPAPAPAPLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGV KALLDEILAALP 259 PRT Artificial ECB147v3LPAPKNLVVSEVTEDSARLSWDDPWAFYE without SFLIQYQESEKVGEAIVLTVPGSERSYDLTGmethionine LCPGTEYTVSIYGVHNVYKDTNMRGLPLS AIFTTAPCPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIV LTVPGSERSYDLTGLCPGTEYVVNIMSVKGGSISPPLSAIFTTAPCPAPAPAPLAEAKVL ANRELDKYGVSDYYKNLINNAKTVEGVK ALLDEILAALP260 PRT Artificial ECB147v4 LPAPKNLVVSEVTEDSARLSWDDPWAFYE withoutSFLIQYQESEKVGEAIVLTVPGSERSYDLTG methionine LKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTED SARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMSV KGGSISPPLSAIFTTAPCPAPAPAPLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGV KALLDEILAALP 261 PRT Artificial ECB147v5LPAPKNLVVSEVTEDSARLSWDDPWAFYE without SFLIQYQESEKVGEAIVLTVPGSERSYDLTGmethionine LCPGTEYTVSIYGVHNVYKDTNMRGLPLS AIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAI VLTVPGSERSYDLTGLCPGTEYVVNIMSVKGGSISPPLSAIFTTAPAPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 262 PRT Artificial ECB147v6 LPAPKNLVVSEVTEDSARLSWDDPWAFYEwithout SFLIQYQESEKVGEAIVLTVPGSERSYDLTG methionineLCPGTEYTVSIYGVHNVYKDTNMRGLPLS AIFTTAPAPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAI VLTVPGSERSYDLTGLKPGTEYVVNIMSVKGGSISPPLSAIFTTAPAPAPAPAPLAEAKV LANRELDKYGVSDYYKNLINNAKTVEGVKALLDEILAALP 263 PRT Artificial ECB147v7 LPAPKNLVVSEVTEDSARLSWDDPWAFYEwithout SFLIQYQESEKVGEAIVLTVPGSERSYDLTG methionineLKPGTEYTVSIYGVHNVYKDTNMRGLPLS AIFTTAPCPAPAPAPLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIRYFEFLGSGEAIV LTVPGSERSYDLTGLKPGTEYVVNIMSVKGGSISPPLSAIFTTAPCPAPAPAPLAEAKVL ANRELDKYGVSDYYKNLINNAKTVEGVK ALLDEILAALP264 PRT Artificial ECB82-cys LPAPKNLVVSEVTEDSARLSWDDPWAFYE withoutSFLIQYQESEKVGEAIVLTVPGSERSYDLTG methionine LKPGTEYTVSIYGVHNVYKDTNMRGLPLSAIFTTAPAPAPAPAPLPAPKNLVVSRVTED SARLSWTAPDAAFDSFWIRYFEFLGSGEAIVLTVPGSERSYDLTGLKPGTEYVVNIMGV KGGKISPPLSAIFTTAPAPAPAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAK TVEGVNALKDEILKAGGHHHHHHC 265 PRTArtificial Tencon-cys LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTG LKPGTEYTVSIYGVKGGHRSNPLSAEFTTG GHHHHHHCCysteine-Engineered EGFR/c-Met Bispecific Molecules (with MethionineIncluded at N-Terminus)

SEQ Cysteine Expression ID Name Position Host Sequence 266 CNTX190- 54(linker in E. Coli MLPAPKNLVVSEVTEDSARLSWDDPWA P54AR4- bold)FYESFLIQYQESEKVGEAIVLTVPGSC 83v22 = EGFR RSYDLTGLKPGTEYTVSIYGVHNVYKDCentyrin TNIRGLPLSAIFTTAPAPAPAPAPLPA P114AR7P95-PKNLVVSRVTEDSARLSWTAPDAAFDS C5v2 FWIRYFEFLGSGEAIVLTVPGSERSYD c-MetCentyrin LTGLKPGTEYVVNILSVKGGSISPPLS Linker AIFTT sequence in bold 267CNTX193- 54 w/H9 E. Coli MLPAPKNLVVSEVTEDSARLSWDDPWA P54AR4- albuminFYESFLIQYQESEKVGEAIVLTVPGSC 83v22 = EGFR bindingRSYDLTGLKPGTEYTVSIYGVHNVYKD Centyrin CentyrinTNIRGLPLSAIFTTAPAPAPAPAPLPA P114AR7P95- (bold/italics)PKNLVVSRVTEDSARLSWTAPDAAFDS C5v2 (linker in bold)FWIRYFEFLGSGEAIVLTVPGSERSYD c-Met Centyrin LTGLKPGTEYVVNILSVKGGSISPPLSLinker AIFTTAPAPAPAPAP

sequence in

bold

268 CNTX194 = 12 (linker in E. Coli MLPAPKNLVVSCVTEDSARLSWDDPWA EGFRbold) FYESFLIQYQESEKVGEAIVLTVPGSE Centyrin RSYDLTGLKPGTEYTVSIYGVHNVYKDP114AR7P95- TNIRGLPLSAIFTTAPAPAPAPAPLPA C5v2 PKNLVVSRVTEDSARLSWTAPDAAFDSc-Met Centyrin FWIRYFEFLGSGEAIVLTVPGSERSYD LinkerLTGLKPGTEYVVNILSVKGGSISPPLS sequence in AIFTT bold 269 CNTX195 = 12 w/H9E. Coli MLPAPKNLVVSCVTEDSARLSWDDPWA EGFR albuminFYESFLIQYQESEKVGEAIVLTVPGSE Centyrin binding RSYDLTGLKPGTEYTVSIYGVHNVYKDP114AR7P95- Centyrin TNIRGLPLSAIFTTAPAPAPAPAPLPA C5v2 (bold/italics)PKNLVVSRVTEDSARLSWTAPDAAFDS c-Met Centyrin (linker in bold)FWIRYFEFLGSGEAIVLTVPGSERSYD Linker LTGLKPGTEYVVNILSVKGGSISPPLS sequencein AIFTTAPAPAPAPAP

bold

270 CNTX196 = 63 (linker in E. Coli MLPAPKNLVVSEVTEDSARLSWDDPWA EGFRbold) FYESFLIQYQESEKVGEAIVLTVPGSE Centyrin RSYDLTGLCPGTEYTVSIYGVHNVYKDP114AR7P95- TNIRGLPLSAIFTTAPAPAPAPAPLPA C5v2 PKNLVVSRVTEDSARLSWTAPDAAFDSc-Met Centyrin FWIRYFEFLGSGEAIVLTVPGSERSYD LinkerLTGLKPGTEYVVNILSVKGGSISPPLS sequence in AIFTT bold 271 CNTX197 = 63 w/H9E. Coli MLPAPKNLVVSEVTEDSARLSWDDPWA EGFR albuminFYESFLIQYQESEKVGEAIVLTVPGSE Centyrin binding RSYDLTGLCPGTEYTVSIYGVHNVYKDP114AR7P95- Centyrin TNIRGLPLSAIFTTAPAPAPAPAPLPA C5v2 (bold/italics)PKNLVVSRVTEDSARLSWTAPDAAFDS c-Met Centyrin (linker in bold)FWIRYFEFLGSGEAIVLTVPGSERSYD Linker LTGLKPGTEYVVNILSVKGGSISPPLS sequencein AIFTTAPAPAPAPAP

bold

272 CNTX198 = C-terminus E. Coli MLPAPKNLVVSEVTEDSARLSWDDPWA EGFR(linker in bold) FYESFLIQYQESEKVGEAIVLTVPGSE CentyrinRSYDLTGLKPGTEYTVSIYGVHNVYKD P114AR7P95- TNIRGLPLSAIFTTAPAPAPAPAPLPA C5v2PKNLVVSRVTEDSARLSWTAPDAAFDS c-Met Centyrin FWIRYFEFLGSGEAIVLTVPGSERSYDLinker LTGLKPGTEYVVNILSVKGGSISPPLS sequence in AIFTTC bold 273 CNTX199 =C-terminus E. Coli MLPAPKNLVVSEVTEDSARLSWDDPWA EGFR w/H9 albuminFYESFLIQYQESEKVGEAIVLTVPGSE Centyrin binding RSYDLTGLKPGTEYTVSIYGVHNVYKDP114AR7P95- Centyrin TNIRGLPLSAIFTTAPAPAPAPAPLPA C5v2 (bold/italics)PKNLVVSRVTEDSARLSWTAPDAAFDS c-Met Centyrin (linker in bold)FWIRYFEFLGSGEAIVLTVPGSERSYD Linker LTGLKPGTEYVVNILSVKGGSISPPLS sequencein AIFTTAPAPAPAPAP

bold

274 Albumin N/A N/A LPAPKNLVVSRVTEDSARLSWTAPDAA bindingFDSFHIEYWEQSIVGEAIVLTVPGSER Centyrin (FN3 SYDLTGLKPGTEYRVWIYGVKGGNDSWdomain) PLSAIFTT

What is claimed:
 1. A protein comprising an amino acid sequence of anyone of SEQ ID NOS: 266-273, wherein the protein has at least onecysteine substitution at a position that corresponds to a positionselected from the group consisting of residues 6, 8, 10, 11, 14, 15, 16,20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89,90, 91, and 93 of the amino acid sequence of SEQ ID NO:
 27. 2. Theprotein of claim 1, wherein the protein is chemically-conjugated to athiol-reactive reagent.
 3. The protein of claim 2, wherein thethiol-reactive reagent is a maleimide moiety.
 4. The protein of claim 3,wherein the maleimide moiety is selected from the group consisting ofNEM, PEG24-maleimide, fluorescein maleimide, MMAE, and MMAF.
 5. Theprotein of claim 1, further comprising a half-life extending moiety. 6.The protein of claim 5, wherein the half-life extending moiety is CD8binding molecule, albumin, an albumin variant, an albumin bindingmolecule, a polyethylene glycol (PEG), CD8, CD8 variant, or at least aportion of an Fc region of an immunoglobulin.
 7. The protein of claim 6,wherein the albumin binding molecule is an albumin binding FN3 domain.8. The protein of claim 1, wherein the protein further comprises theamino acid sequence of SEQ ID NO:274.
 9. A protein comprising an aminoacid sequence selected from the group consisting of SEQ ID NO:266, SEQID NO:267, SEQ ID NO:268, SEQ ID NO:269, SEQ ID NO:270, SEQ ID NO:271,and SEQ ID NO:273.
 10. An isolated polynucleotide encoding the proteinof claim
 1. 11. A vector comprising the polynucleotide of claim
 10. 12.An isolated host cell comprising the vector of claim
 11. 13. A method ofproducing the protein according to claim 1, wherein a cell comprising apolynucleotide encoding the protein is cultured under conditions toproduce the protein.
 14. The method of claim 13, further comprisingintroducing the polynucleotide encoding the protein into the cell priorto the culturing step.
 15. The method of claim 13, further comprisingpurifying the protein.